Connective tissue growth factor antibodies

ABSTRACT

The present invention relates to antibodies that bind to CTGF. The antibodies are particularly directed to regions of CTGF involved in biological activities associated with fibrosis. The invention also relates to methods of using the antibodies to treat disorders associated with CTGF including localized and systemic fibrotic disorders including those of the lung, liver, heart, skin, and kidney.

This application is a continuation of U.S. application Ser. No.16/156,873, filed on 10 Oct. 2018, which is a continuation of U.S.application Ser. No. 15/164,615, filed on 25 May 2016, now abandoned,which is a continuation of U.S. application Ser. No. 14/690,727, filed20 Apr. 2015, now abandoned, which is a continuation of U.S. applicationSer. No. 13/691,356, filed 30 Nov. 2012, now U.S. Pat. No. 9,034,643,which is a continuation of U.S. application Ser. No. 12/927,243, filed10 Nov. 2010, now abandoned, which is a continuation of U.S. applicationSer. No. 12/157,262, filed 9 Jun. 2008, now U.S. Pat. No. 7,871,617,which is a continuation of U.S. application Ser. No. 10/858,186, filed 1Jun. 2004, now U.S. Pat. No. 7,405,274, which claims the benefit of U.S.Provisional Application Ser. No. 60/475,598, filed on 4 Jun. 2003,incorporated in their entireties by reference herein.

FIELD OF THE INVENTION

The present invention relates to antibodies that bind to connectivetissue growth factor (CTGF). The antibodies are particularly directed toregions of CTGF involved in biological activities associated withvarious disorders.

BACKGROUND Connective Tissue Growth Factor (CTGF)

CTGF is a 36 kD, cysteine-rich, heparin binding, secreted glycoproteinoriginally isolated from the culture media of human umbilical veinendothelial cells. (See e.g., Bradham et al. (1991) J Cell Biol114:1285-1294; Grotendorst and Bradham, U.S. Pat. No. 5,408,040.) CTGFbelongs to the CCN (CTGF, Cyr61, Nov) family of proteins (secretedglycoproteins), which includes the serum-induced immediate early geneproduct Cyr61, the putative oncogene Nov, the ECM-associated proteinFISP-12, the src-inducible gene CEF-10, the Wnt-inducible secretedprotein WISP-3, and the anti-proliferative protein HICP/rCOP (Brigstock(1999) Endocr Rev 20:189-206; O'Brian et al. (1990) Mol Cell Biol10:3569-3577; Joliot et al. (1992) Mol Cell Biol 12:10-21; Ryseck et al.(1990) Cell Growth and Diff 2:225-233; Simmons et al. (1989) Proc NatlAcad Sci USA 86:1178-1182; Pennica et al. (1998) Proc Natl Acad Sci USA,95:14717-14722; and Zhang et al. (1998) Mol Cell Biol 18:6131-6141.) CCNproteins are characterized by conservation of 38 cysteine residues thatconstitute over 10% of the total amino acid content and give rise to amodular structure with N- and C-terminal domains. The modular structureof CTGF includes conserved motifs for insulin-like growth factor bindingprotein (IGF-BP) and von Willebrand's factor (VWC) in the N-terminaldomain, and thrombospondin (TSP1) and a cysteine-knot motif in theC-terminal domain.

CTGF expression is induced by members of the Transforming Growth Factorbeta (TGFβ) superfamily, which includes TGFβ-1, -2, and -3, bonemorphogenetic protein (BMP)-2, and activin, as well as a variety ofother regulatory modulators including dexamethasone, thrombin, vascularendothelial growth factor (VEGF), and angiotensin II; and environmentalstimuli including hyperglycemia and hypertension. (See, e.g., Franklin(1997) Int J Biochem Cell Biol 29:79-89; Wunderlich (2000) Graefes ArchClin Exp Ophthalmol 238:910-915; Denton and Abraham (2001) Curr OpinRheumatol 13:505-511; and Riewald (2001) Blood 97:3109-3116; Riser etal. (2000) J Am Soc Nephrol 11:25-38; and International Publication No.WO 00/13706). TGFβ stimulation of CTGF expression is rapid andprolonged, and does not require persistent application. (Igarashi et al.(1993) Mol Biol Cell 4: 637-645.) Enhanced expression of CTGF by TGFβinvolves transcriptional activation via DNA regulatory elements presentin the CTGF promoter. (Grotendorst et al. (1996) Cell Growth Differ 7:469-480; Grotendorst and Bradham, U.S. Pat. No. 6,069,006; Holmes et al.(2001) J Biol Chem 276:10594-10601.)

CTGF has been shown to increase steady-state transcription of α1(I)collagen, α5 integrin, and fibronectin mRNAs, as well as to promotecellular processes including proliferation and chemotaxis of variouscell types in culture. (See e.g., Frazier et al. (1996) J InvestDermatol 107:406-411; Shi-wen et al. (2000) Exp Cell Res 259:213-224;Klagsburn (1977) Exp Cell Res 105:99-108; Gupta et al. (2000) Kidney Int58:1389-1399; Wahab et al. (2001) Biochem J 359(Pt 1):77-87; Uzel et al.(2001) J Periodontol 72:921-931; and Riser and Cortes (2001) Ren Fail23:459-470.) Subcutaneous injection of CTGF in neonatal mice results inthe local deposition of granulation tissue. Similarly, subcutaneousinjection of TGFβ generates granulation tissue formation and induceshigh levels of CTGF mRNA in local fibroblasts. Moreover, combination orsequential treatment with TGFβ and CTGF results in the development of amore persistent granuloma. (Mori et al. (1999) J Cell Physiol181:153-159.) Thus, CTGF appears to mediate a subset of the effectselicited by TGFβ, in particular, the production and deposition ofextracellular matrix (ECM). Further, the ability to respond to CTGF, orthe extent of the CTGF response, may rely upon a priming stimulusprovided by TGFβ treatment that enables cellular “competence.”(International Publication No. WO 96/08140.)

Although a plethora of interacting factors have been characterized thatmodulate tissue organization, a consensus is now emerging for the roleof CTGF in regulating skeletal development, wound healing andextracellular matrix (ECM) remodeling, fibrosis, tumorigenesis, andangiogenesis. For example, elevated CTGF expression has been observed incirrhotic liver, pulmonary fibrosis, inflammatory bowel disease,sclerotic skin and keloids, desmoplasia, and atherosclerotic plaques.(Abraham et al. (2000) J Biol Chem 275:15220-15225; Dammeier et al.(1998) Int J Biochem Cell Biol 30:909-922; diMola et al. (1999) Ann Surg230(1):63-71; Igarashi et al. (1996) J Invest Dermatol 106:729-733; Itoet al. (1998) Kidney Int 53:853-861; Williams et al. (2000) J Hepatol32:754-761; Clarkson et al. (1999) Curr Opin Nephrol Hypertens8:543-548; Hinton et al. (2002) Eye 16:422-428; Gupta et al. (2000)Kidney Int 58:1389-1399; Riser et al. (2000) J Am Soc Nephrol 11:25-38.)

CTGF is also upregulated in glomerulonephritis, IgA nephropathy, focaland segmental glomerulosclerosis and diabetic nephropathy. (See, e.g.,Riser et al. (2000) J Am Soc Nephrol 11:25-38.) An increase in thenumber of cells expressing CTGF is also observed at sites of chronictubulointerstitial damage, and CTGF levels correlate with the degree ofdamage. (Ito et al. (1998) Kidney Int 53:853-861.) Further, CTGFexpression is increased in the glomeruli and tubulointerstium in avariety of renal diseases in association with scarring and sclerosis ofrenal parenchyma. Elevated levels of CTGF have also been associated withliver fibrosis, myocardial infarction, and pulmonary fibrosis. Forexample, in patients with idiopathic pulmonary fibrosis (IPF), CTGF isstrongly upregulated in biopsies and bronchoalveolar lavage fluid cells.(Ujike et al. (2000) Biochem Biophys Res Commun 277:448-454; Abou-Shadyet al. (2000) Liver 20:296-304; Williams et al. (2000) J Hepatol32:754-761; Ohnishi et al. (1998) J Mol Cell Cardiol 30:2411-22; Laskyet al. (1998) Am J Physiol 275: L365-371; Pan et al. (2001) Eur Respir J17:1220-1227; and Allen et al. (1999) Am J Respir Cell Mol Biol21:693-700.) Thus, CTGF represents a valid therapeutic target indisorders, such as those described above.

The association of CTGF with various aspects of these disorders has beenestablished; and methods for treating disorders through modulation ofCTGF have been described. (See, e.g., Grotendorst and Bradham, U.S. Pat.No. 5,783,187; International Publication No. WO 00/13706; andInternational Publication No. WO 03/049773.) Modulation of growthfactors, cytokines, and cell surface receptors can be accomplished usingmonoclonal antibodies, and several therapeutic monoclonal antibodieshave been approved or are underdevelopment. (See, e.g., Infliximab(Remicade; Maini et al. (1998) Arthritis Rheum 41:1552-1563; Targan etal. (1997) N Engl J Med 337:1029-1035); Basiliximab (Simulect) andDaclizumab (Zenapax) (Bumgardner et al. (2001) Transplantation72:839-845; Kovarik et al. (1999) Transplantation 68:1288-1294); andTrastuzumab (Herceptin; Baselga (2001) Ann Oncol 12 Suppl 1:S49-55.))

Antibodies have been generated against CTGF, and have proven efficaciousin vivo at, e.g., inhibiting angiogenesis. (See, e.g., Grotendorst andBradham, U.S. Pat. No. 5,408,040; International Publication No. WO99/07407; and Shimo et al. (2001) Oncology 61:315-322). Further, themodular nature of CTGF appears to distinguish domains involved inspecific biological activities. For example, the N-terminal half of CTGFhas been shown to stimulate cell differentiation and ECM production,whereas the C-terminal half stimulates cell proliferation. (See, e.g.,International Publication Nos. WO 00/35936 and WO 00/35939; andBrigstock and Harding, U.S. Pat. No. 5,876,70.) This demonstrates thatantibodies directed to different regions of the CTGF molecule exhibitdifferent effects with respect to modulating biological activities ofCTGF. (See, e.g., International Publication Nos. WO 00/35936 and WO00/35939). Currently, no clear distinction has been made betweenanti-CTGF antibodies that produce a desired effect, and those whicheither produce multiple effects or are non-neutralizing. (See, e.g.,International Publication No. WO 99/33878.)

There is clearly a need in the art for agents that effectivelyneutralize the activity of CTGF in disease. Antibodies, particularlymonoclonal antibodies, provide the specificity and pharmacokineticprofiles appropriate for a therapeutic agent, and neutralizingantibodies targeted to specific activities of CTGF would fulfill a needin the art and would find use in therapeutic treatment ofCTGF-associated disorders including pulmonary disorders such asidiopathic pulmonary fibrosis (IPF), etc.; renal disorders such asdiabetic nephropathy, glomerulosclerosis, etc.; and ocular disorderssuch as retinopathy, macular degeneration, etc.

SUMMARY OF THE INVENTION

The present invention provides antibodies, particularly monoclonalantibodies, and portions thereof that specifically bind to a region onthe N-terminal fragment of a CTGF polypeptide.

In one aspect, an antibody of the invention specifically binds to aregion on human CTGF (SEQ ID NO:2) as set forth from about amino acid103 to amino acid 164 (SEQ ID NO:21), more specifically from about aminoacid 135 to about amino acid 157 (SEQ ID NO:22); and even morespecifically from about amino acid 142 to about amino acid 154 (SEQ IDNO:25); or an orthologous region on CTGF derived from another species.In particular embodiments, the antibody has the same specificity as anantibody produced by the cell line identified by ATCC Accession No.PTA-6006. (Deposited with the ATCC on 20 May 2004.) In specificembodiments, the antibody is substantially identical to mAb1, asdescribed infra. More preferably, the antibody is substantially similarto CLN1, as described infra. In yet another embodiment, an antibody ofthe invention competitively binds with any of the foregoing antibodiesto a CTGF polypeptide.

In one embodiment, the present invention provides a monoclonal antibodyor portion thereof comprising at least one member of the groupconsisting of an immunoglobulin heavy chain sequence comprising SEQ IDNO:14, an immunoglobulin heavy chain sequence comprising the variabledomain of SEQ ID NO:14, an immunoglobulin light chain sequencecomprising SEQ ID NO:20, an immunoglobulin light chain sequencecomprising the variable domain of SEQ ID NO:20, or conservative variantsthereof. In a specific embodiment, the antibody comprises theimmunoglobulin heavy chain variable domain from amino acid residue 1through amino acid residue 167 of SEQ ID NO:14. In another specificembodiment, the antibody comprises the immunoglobulin light chainvariable domain from amino acid residue 1 through amino acid residue 136of SEQ ID NO:20. In a particular embodiment, the antibody comprises theimmunoglobulin heavy chain sequence of SEQ ID NO:14 and theimmunoglobulin light chain sequence of SEQ ID NO:20. Within thisembodiment, the present invention specifically provides the antibody ofCLN1 or a portion thereof comprising at least the antigen binding regionresidues of CLN1.

In certain aspects, the antibody of the invention is a polyclonalantibody. In other aspects, the antibody is a monoclonal antibody. Incertain embodiments, the antibody is a humanized monoclonal antibody;more preferably a human monoclonal antibody. Any of the aforementionedantibodies may additionally contain various amounts of glycosylation,incorporated by the cell producing the antibody or applied and/ormodified synthetically; or the antibody may be free of glycosylation.The antibody may be optionally pegylated and/or similarly modified toincrease plasma half-life, etc. In various embodiments, the inventionprovides fragments of the antibody, particularly wherein the fragment isa Fab, F(ab)₂, or Fv fragment.

In certain aspects, the antibody or portion thereof is produced by acloned cell line. The cell line may be derived from any animal modelused for monoclonal antibody production including, but not limited to,mice, goat, chicken, etc. In particular, the cell line may be derivedfrom mice. The mice may be standard mice used for antibody production,e.g., BALB/C, or a modified, e.g., transgenic, mouse strain optimized ordeveloped for production of specific isotype, idiotype, orspecies-specific monoclonal antibodies. In one embodiment, the cell lineis a hybridoma cell line that produces and secretes mAb1. In otherembodiments, the cell line produces and secretes an antibody or portionthereof that has a property substantially equivalent to mAb1. In stillother embodiments, the cell line produces and secretes an antibody orportion thereof that has a property substantially equivalent to CLN1. Ina particular embodiment, the invention provides a cell line identifiedby ATCC Accession No. PTA-6006. (Deposited 20 May 2004.)

In another aspect, the antibody or portion thereof is derived from anon-human transgenic animal, particularly a non-human transgenic mammal,capable of producing a human antibody. The animal may be of any speciesincluding, but not limited to, mouse, chicken, cow, goat, etc. Inparticular, the animal may be mouse. Such antibodies may be obtainableby immunizing a non-human transgenic mammal with a fragment of humanCTGF, e.g., SEQ ID NO:21, or, more specifically, SEQ ID NO:22, or to anorthologous region on CTGF derived from a non-human species. In certainembodiments, the antibodies are obtained by immunizing the non-humantransgenic mammal with a fragment of CTGF selected from the groupconsisting of SEQ ID NOs:23 thru 26 or an orthologous region on CTGFderived from a non-human species. In specific embodiments, theantibodies are obtained by immunizing a transgenic mouse with any of theaforementioned CTGF fragments. In other embodiments, the antibodies areobtained by immunizing a transgenic mouse with functional equivalents ofany of the aforementioned CTGF fragments.

By “specifically binds to a region on CTGF”, it is meant that theantibodies have binding specificity for a particular region on CTGF,which may be defined by a linear amino acid sequence, or by a tertiary,i.e., three-dimensional, conformation on part of the CTGF polypeptide.Binding specificity means that the antibodies affinity for the portionof CTGF is substantially greater than their affinity for other relatedpolypeptides. By “substantially greater affinity” we mean that there isa measurable increase in the affinity for the portion of CTGF ascompared with the affinity for other related polypeptides. Preferably,the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold,10³-fold, 10⁴-fold, 10⁵-fold, 10⁶-fold or greater for the particularportion of CTGF than for other proteins. Preferably, the bindingspecificity is determined by affinity chromatography,immunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA),or by fluorescence-activated cell sorting (FACS) analysis. Morepreferably, the binding specificity is obtained by RIA or affinitychromatography, as described infra.

In preferred embodiments of the invention, the antibodies have anaffinity that is equal to or greater than that of mAb1, described infra,as determined, for example, by the Scatchard analysis of Munson andPollard (1980, Anal Biochem 107:220). Antibody affinity is defined asthe strength of the total noncovalent interactions between a singleantigen-binding site on an antibody and a single epitope on an antigen.Affinity is calculated by measuring the association constant (K_(a)),such that

${Affinity} = {K_{a} = {\frac{\left\lbrack {{Ab} \cdot {Ag}} \right\rbrack}{\left\lbrack {Ab} \right\rbrack\left\lbrack {Ag} \right\rbrack} = \frac{1}{K_{d}}}}$

where [Ab] is the concentration of free antigen binding site on theantibody, [Ag] is the concentration of free antigen, [Ab·Ag] is theconcentration of antigen binding site on the antibody occupied byantigen, and K_(d) is the dissociation constant of the antibody-antigencomplex. Preferably, antibodies of the invention have an affinity forCTGF that is greater than Kd=10⁻⁸, preferably greater than 10⁻⁹,preferably greater than 10⁻¹⁰, particularly for therapeutic use.Advantageously, an antibody according to the invention has an affinitysimilar to or greater than that of mAb1 (that is, a Kd≤10⁻⁹). However,antibodies sharing epitope binding with mAb1, but having lower affinity(i.e., higher Kd) than mAb1, are also embodied within the presentinvention and are potentially useful in various assays and diagnosticapplications as described herein. Such antibodies may additionally beuseful in therapeutic applications, especially if they have a highavidity for antigen, as described below.

Antibodies according to the invention may be monovalent, bivalent orthey may be multi-valent. In certain embodiments of the invention, it ispreferred that the antibodies of the invention are bivalent ormultivalent. Any of the antibodies of the invention may be manipulatedto improve avidity, e.g., by combining epitope-binding sites into asingle antibody construct, e.g., a tribody, etc. Antibodies according tothe invention may be single chain antibodies.

It may be useful in some circumstances for antibodies of the inventionto show suitable affinity for CTGF from other species, for example, fortreatment and prevention of disorders in those species. For example, anantibody of the invention that shows a suitable K_(d) for canine CTGFcould be used to treat a CTGF-associated disorder in dogs. Antibodies ofthe invention that show cross-species affinity, such as mAb1, are alsouseful as research tools, to study CTGF-associated disorders in variousanimal models. In another aspect, the antibody or portion thereof isencoded by genetic material originally derived from a human. Theantibody may be generated by cells in culture, e.g., using phage displaytechniques, or may be produced within an animal, e.g., a non-humantransgenic animal containing immunoglobulin genes derived from a human.

Additionally, the invention provides recombinant constructs comprisingportions of any of the antibodies of the invention, as described above,and a protein derived from another source. Specifically contemplated areembodiments encompassing chimeric antibodies comprising a variableregion derived from a monoclonal antibody that specifically binds to aregion on an N-terminal fragment of CTGF and a constant region derivedfrom another source. The variable region can be derived from anyantibody defined by the invention, and specifically encompassesantibodies that bind to a region on human CTGF from about amino acid 97to about amino acid 180 of SEQ ID NO:2, or, more specifically, fromabout amino acid 103 to about amino acid 164 of SEQ ID NO:2, or, morespecifically, from about amino acid 134 to about amino acid 158 of SEQID NO:2, or, even more specifically, from about amino acid 143 to aboutamino acid 154 of SEQ ID NO:2, or to an orthologous region on CTGFderived from another species. The constant region can be derived fromany source. In some embodiments, the constant region is derived from aconstant region of a human immunoglobulin.

The present invention also provides any of the antibodies describedabove wherein the antibody additionally comprises a labeling agentcapable of providing a detectable signal by itself or together withother substances. Such labeling agents can be selected from, but are notlimited to, the group consisting of an enzyme, fluorescent substance,chemiluminescent substance, biotin, avidin, and radioisotope. Thepresent invention also provides any of the antibodies described abovewherein the antibody additionally comprises a cytotoxic agent or enzyme.

In other embodiments, the antibodies of the invention, as describedabove, additionally neutralize at least one activity associated withCTGF. Such activities associated with CTGF include, but are not limitedto, stimulation of cell migration, production of extracellular matrix bya cell in vivo or ex vivo, and/or reduction in fibrosis in a subject. Inparticular embodiments, the biological activity is selected from thegroup consisting of cell growth, differentiation of fibroblasts and/orendothelial cells, and induction of expression of proteins involved inextracellular matrix formation and remodeling including, e.g., collagensincluding, but not limited to, types I, II, III, and IV; andfibronectin.

In certain embodiments, the antibodies specifically inhibit cellmigration in ex vivo assays. Preferably, the antibodies inhibitCTGF-stimulated chemotactic migration of smooth muscle cells in a Boydenchamber assay. For example, in a cell migration assay described infra,antibodies of the invention repeatedly and reproducibly inhibitCTGF-induced migration. In various embodiments, the antibodiesspecifically reduce fibrosis in animal models. Preferably, theantibodies inhibit development of fibrosis in animal models of lung andkidney fibrosis. For example, the antibodies attenuate bleomycin-inducedlung fibrosis in mice by 60-70%, as determined by inhibition ofpulmonary hydroxyproline (collagen) accumulation and/or histologicalexamination of tissue preparations, described infra. Further, theantibodies reduce the accumulation of collagen in a rat remnant kidney(i.e., 5/6 nephrectomy) model, and in mice following unilateral ureterobstruction (UUO), as described infra.

In other embodiments, antibodies of the invention modulate theinteraction between a CTGF polypeptide and a cell receptor, and/orbetween a CTGF polypeptide and a secreted or membrane-associatedcofactor, thereby neutralizing a biological activity of CTGF. Thecofactor may be any protein, carbohydrate, and/or lipid; in particularembodiments, the cofactor is a member of the TGF-β family of growthfactors, e.g., TGF-β, BMP-4, etc.

In another aspect, the antibody reduces fibrosis in a subject. Invarious embodiments, the subject is a tissue or organ. In otherembodiments, the subject is an animal, preferably a mammal, mostpreferably a human. When the subject is a tissue, the inventionspecifically contemplates both endogenous tissues and ex vivo tissues,e.g., transplant tissues, tissues grown in culture, etc. In variousembodiments, the tissue is selected from the group consisting ofepithelial, endothelial, and connective tissue. When the subject is anorgan, the invention specifically contemplates organs selected from thegroup consisting of kidney, lung, liver, eye, heart, and skin. Inpreferred embodiments, the subject is an animal, particularly, an animalof mammalian species including rat, rabbit, bovine, ovine, porcine,murine, equine, and primate species. In a most preferred embodiment, thesubject is human.

In specific embodiments, the antibody is used to treat or prevent aCTGF-associated disorder in a subject having or at risk for having aCTGF-associated disorder. Such disorders include, but are not limitedto, various cancers including acute lymphoblastic leukemia,dermatofibromas, breast cancer, breast carcinoma, glioma andglioblastoma, rhabdomyosarcoma and fibrosarcoma, desmoplasia,angiolipoma, angioleiomyoma, desmoplastic cancers, and prostate,ovarian, colorectal, pancreatic, gastrointestinal, and liver cancer andother tumor growth and metastases. CTGF-associated disorders alsoinclude various fibrotic disorders including, but not limited to,idiopathic pulmonary fibrosis, kidney fibrosis, glomerulosclerosis,ocular fibrosis, osteoarthritis, scleroderma, cardiac fibrosis, andliver fibrosis. Fibrosis can occur in any organ or tissue including anorgan selected from, but not limited to, kidney, lung, liver, heart, andskin; or a tissue selected from, but not limited to, epithelial,endothelial, and connective tissue. In other embodiments, theCTGF-associated disorder may be caused by any initiating factorincluding, but not limited to, exposure to chemicals or biologicalagents, inflammatory response, autoimmune reaction, trauma, surgicalprocedures, etc. CTGF-associated disorders also include, but are notlimited to, disorders due to hyperglycemia and hypertension. Suchdisorders may occur, e.g., due to diabetes, obesity, etc., and includediabetic nephropathy, retinopathy, and cardiovascular disease.

Therefore, in various embodiments, the invention provides antibodiesthat can be used to treat or prevent CTGF-associated disorders in asubject. The present invention also provides the use of such antibodiesin the manufacture of a medicament for the treatment of CTGF-associateddisorders.

In another aspect, the invention provides a method of neutralizing anactivity associated with CTGF comprising contacting an antibody of theinvention and a CTGF polypeptide, thereby neutralizing a biologicalactivity of CTGF, such as those described above. The biological activitycan be any activity of CTGF including, but not limited to, stimulationof cell migration and production of extracellular matrix. In variousembodiments, the neutralizing occurs in vitro. In other embodiments, theneutralizing occurs in a subject in vivo.

In yet another aspect, the invention provides methods of using anantibody as described above to treat a CTGF-associated disorder in apatient in need, the method comprising administering the antibody or apharmaceutical formulation thereof to the patient, thereby treating thedisorder. The subject can be a patient diagnosed with or suspected ofhaving a CTGF-associated disorder, including, e.g., a disorder resultingin excess production of extracellular matrix. In particular aspects,CTGF-associated disorder is selected from a cancer or fibrotic disorder.Cancers include, but are not limited to, acute lymphoblastic leukemia,dermatofibromas, breast cancer, breast carcinoma, glioma andglioblastoma, rhabdomyosarcoma and fibrosarcoma, desmoplasia,angiolipoma, angioleiomyoma, desmoplastic cancers, and prostate,ovarian, colorectal, pancreatic, gastrointestinal, and liver cancer, andfibrotic disorders include, but are not limited to, idiopathic pulmonaryfibrosis, kidney fibrosis, glomerulosclerosis, ocular fibrosis, maculardegeneration, osteoarthritis, scleroderma, chronic heart failure,cardiac fibrosis, and liver fibrosis. In other embodiments, theCTGF-associated disorder may be caused by any initiating factorincluding, but not limited to, exposure to chemicals or biologicalagents, inflammatory response, autoimmune reaction, trauma, surgicalprocedures, etc. CTGF-associated disorders also include, but are notlimited to, disorders due to hyperglycemia and hypertension. Suchdisorders may occur, e.g., due to diabetes, obesity, etc., and includediabetic nephropathy, retinopathy, and cardiovascular disease.

In another aspect, the present invention provides a compositioncomprising an antibody as described above and at least one othercomponent. Components may include any compound, molecule, or agent,including, e.g., proteins, nucleic acids, carbohydrates, lipids, etc.Additionally, components may include various solvents, salts, and othercarriers and/or excipients. In some embodiments, the composition is apharmaceutical composition comprising an antibody as described above andat least one additional component selected from a solvent, a stabilizer,or an excipient. In a particular embodiment, the pharmaceuticalcomposition comprises an antibody in admixture with a pharmaceuticallyacceptable carrier. The pharmaceutical composition may additionallycontain a second therapeutic agent, e.g., an angiotensin convertingenzyme (ACE) inhibitor, an advanced glycation endproduct cleavage orinhibitory agent, etc. The invention additionally provides medicamentscomprising an antibody as defined above for treating a subject having aCTGF-associated disorder. Such disorders include, but are not limitedto, various cancers and fibrotic disorders; disorders resulting fromconditions such as myocardial infarction, arthritis, and inflammation;and disorders due to diabetes, obesity, and the like, which may includediabetic nephropathy, retinopathy, and cardiovascular disease.

In another embodiment, the invention provides a polypeptide sequenceselected from the group consisting of SEQ ID NO:14, amino acid 1 throughamino acid 167 of SEQ ID NO:14, SEQ ID NO:20, and amino acid 1 throughamino acid 136 of SEQ ID NO:20. The invention also encompassesconservative variants of the polypeptides. In another embodiment, theinvention provides specific fragments of human CTGF selected from thegroup consisting of SEQ ID NOs:21 through 26, and orthologous CTGFfragments obtained from a non-human species.

The polypeptides referred to above may be “altered” polypeptides, asdefined infra.

In another embodiment, the invention provides a polynucleotide sequenceencoding an antibody of the invention or a portion thereof. Inparticular embodiments, the polynucleotide sequence is selected from thegroup consisting of a polynucleotide sequence encoding SEQ ID NO:14, apolynucleotide sequence encoding from amino acid 1 through amino acid167 of SEQ ID NO:14, the polynucleotide sequence of SEQ ID NO:13, and apolynucleotide comprising nucleotide 1 through nucleotide 501 of SEQ IDNO:13. In other embodiments, the polynucleotide sequence is selectedfrom the group consisting of a polynucleotide sequence encoding SEQ IDNO:20, a polynucleotide sequence encoding from amino acid 1 throughamino acid 136 of SEQ ID NO:20, the polynucleotide of SEQ ID NO:19, anda polynucleotide comprising nucleotide 1 through nucleotide 408 of SEQID NO:19.

The polynucleotides referred to above may be “altered” polynucleotides,as defined infra.

The invention additionally provides recombinant polynucleotidescomprising any of the polynucleotide sequences described above operablylinked to a vector sequence that contains replication andtranscriptional control sequences. In one aspect, the recombinantpolynucleotide encodes the amino acid sequence of SEQ ID NO:14 or thevariable domain therein. In another aspect, the recombinantpolynucleotide comprises SEQ ID NO:13. In yet another aspect, therecombinant polynucleotide encodes the amino acid sequence of SEQ IDNO:20 or the variable domain therein. In still another aspect, therecombinant polynucleotide comprises SEQ ID NO:19.

The invention also provides host cells transfected with at least one ofthe recombinant polynucleotides described above. Host cells include anyprokaryotic and eukaryotic host cell, including, e.g., cloned cell linesmaintained by culture methods known to those of skill in the art. Hostcells also include transgenic plants and animals derived fromtransformed cells, e.g., stem cells. In one embodiment, the host cellcomprises a cell co-transfected with a polynucleotide encoding SEQ IDNO:14 and a polynucleotide encoding SEQ ID NO:20, and which produces afunctional antibody with characteristics substantially the same as mAb1.In particular embodiments, the antibody is CLN1. In another particularembodiment, the host cell is identified by ATCC Accession No. PTA-6006.(Deposited: 20 May 2004.)

These and other embodiments of the subject invention will readily occurto those of skill in the art in light of the disclosure herein, and allsuch embodiments are specifically contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the structure and sequence conservation ofConnective Tissue Growth Factor. FIG. 1A shows the modular domainstructure of CTGF, which includes conserved motifs for insulin-likegrowth factor binding protein (IGF-BP) and Von Willebrand's factor (VWC)in the N-terminal fragment, and thrombospondin (TSP1) and thecysteine-knot motif (CT) in the C-terminal fragment. FIG. 1B shows amultiple sequence alignment between N-terminal fragments of human(hCTGF; SEQ ID NO:27), bovine (bCTGF; SEQ ID NO:29), porcine (pCTGF; SEQID NO:30), rat (rCTGF; SEQ ID NO:31), and murine (FISP12; SEQ ID NO:32)CTGF orthologs. The alignment was created using the CLUSTAL W program(v. 1.74; Thompson et al. (1994) Nucleic Acids Res 22:4673-4680) usingdefault parameters. In the figure, an asterisk (*) indicates completeconservation of the amino acid residue among the species represented.

FIGS. 2A and 2B show Scatchard plots of competitive binding betweenlabeled and unlabeled human CTGF to anti-CTGF antibodies, mAb2 and mAb1,respectively. mAb1 is an exemplary antibody of the present invention.

FIG. 3A shows Fab antibody fragment (M_(r) 45 kD) obtained followingpapain digestion of the corresponding IgG antibody mAb1 and subsequentprotein A-Sepharose affinity chromatography (Lane 2), as demonstrated bySDS-PAGE. FIG. 3B shows binding of a Fab fragment and corresponding IgGto CTGF over increasing concentration of chaotropic agent (thiocyanate).

FIGS. 4A and 4B show Scatchard plots of competitive binding betweenlabeled recombinant human CTGF and unlabeled rat CTGF to anti-CTGFantibodies, mAb2 and mAb1, respectively.

FIGS. 5A, 5B, and 5C show the therapeutic benefits of an antibody of theinvention in a model of interstitial fibrosis in the lung. FIG. 5A showsthe effect of antibody treatment on bleomycin-induced increase inhydroxyproline content of mouse lungs. The number of animals in eachgroup is shown in parentheses below each bar and treatment groups areindicated along the x-axis. SA: Saline; BL: Bleomycin; AbsJ: pool of 3monoclonal antibodies of the invention; mAb1, an exemplary antibody ofthe invention. Values are expressed as mean±SE. FIGS. 5B and 5C showhematoxylin- and eosin-stained paraffin sections of pulmonary proximalacini from mice exposed to bleomycin by intratracheal injection andsubsequently treated with saline or antibodies of the invention,respectively. In FIG. 5B, the thin interalveolar septa acinus have anabnormal appearance, and inflammatory cells and fibrosis are present. InFIG. 5C, the parenchyma is largely normal and there is only moderatethickening of interalveolar septa.

FIGS. 6A, 6B, and 6C show the therapeutic benefits of an antibody of theinvention in a model of tubulointerstitial fibrosis in the kidney. FIG.6A shows the reduction in fibrosis due to unilateral ureter obstruction(UUO) following treatment with an antibody of the invention, mAb1, or anantibody directed to the C-terminus of CTGF, mAb3. The extent offibrosis is expressed as the ratio of hydroxyproline to proline in theobstructed kidney compared with the contralateral unobstructed kidney(mean±SE). FIGS. 6B and 6C show trichome-stained paraffin sections ofobstructed kidneys receiving saline or antibody therapy, respectively.

FIGS. 7A and 7B show the therapeutic benefit of an antibody of theinvention in a model of glomerular fibrosis in the kidney. FIGS. 7A and7B show photomicrographs of trichrome-stained remnant kidney tissueafter receiving saline or antibody therapy, respectively.

FIGS. 8A, 8B, 8C, 8D, 8E, 8F, and 8G show the induction of localizedsubcutaneous granulomas in newborn mice. On the left, FIGS. 8A and 8Bshow the formation of granuloma at the site of subcutaneous injection ofTGFβ alone or TGFβ and CTGF, respectively. On the right, FIGS. 8Cthrough 8G show a histological panel representing the scoring system(from 0 [normal] to 4 [fibrotic]) used to evaluate therapeutic benefitof antibody.

FIG. 9 shows degree of fibrosis in a localized subcutaneous granulomamodel with and without treatment with anti-CTGF antibodies. mAb1 is anexemplary antibody of the invention, whereas mAb3 is an anti-CTGFantibody that specifically binds to a C-terminal CTGF epitope.

FIGS. 10A, 10B, 10C, and 10D show the therapeutic benefit of an antibodyof the invention in organ fibrosis using a model of systemic sclerosis.Each of the panels shows changes in collagen accumulation in respectiveorgans following treatment with saline (control), TGFβ and CTGF, or TGFβand CTGF treatment concomitant with antibody therapy.

FIGS. 11A and 11B show a diagramatic representation of the cloning ofheavy and light immunoglobulin chains of an exemplary antibody of theinvention, mAb1. FIG. 11A shows the alignment of heavy chain PCRfragments used to determine the mAb1 heavy chain coding sequence (CDS).FIG. 11B shows the alignment of light chain PCR fragments used todetermine the mAb1 light chain coding sequence (CDS).

FIGS. 12A and 12B shows binding studies between CTGF and TGFβ. FIG. 12Ashows the degree of binding between TGFβ and either CTGF, a fragment ofCTGF encoded by exon 3 (Exon 3), or a fragment of CTGF encoded by exon 5(Exon 5) in the presence or absence of anti-CTGF antibody. FIG. 12Bshows the degree to which anti-CTGF antibodies inhibit TGFβ and CTGFinteraction. In the figure, antibodies include exemplary antibodies ofthe invention, mAb1 and mAb4, and an antibody that specifically binds toa C-terminal CTGF epitope, mAb3.

DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that the invention is not limited to the particularmethodologies, protocols, cell lines, assays, and reagents described, asthese may vary. It is also to be understood that the terminology usedherein is intended to describe particular embodiments of the presentinvention, and is in no way intended to limit the scope of the presentinvention as set forth in the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural references unlesscontext clearly dictates otherwise. Thus, for example, a reference to “afragment” includes a plurality of such fragments, a reference to an“antibody” is a reference to one or more antibodies and to equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications cited hereinare incorporated herein by reference in their entirety for the purposeof describing and disclosing the methodologies, reagents, and toolsreported in the publications, which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, cell biology, genetics, immunology and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Gennaro, A. R., ed. (1990) Remington's PharmaceuticalSciences, 18th ed., Mack Publishing Co.; Colowick, S. et al., eds.,Methods In Enzymology, Academic Press, Inc.; Handbook of ExperimentalImmunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986,Blackwell Scientific Publications); Maniatis, T. et al., eds. (1989)Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. Cold SpringHarbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) ShortProtocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream etal., eds. (1998) Molecular Biology Techniques: An Intensive LaboratoryCourse, Academic Press); PCR (Introduction to Biotechniques Series), 2nded. (Newton & Graham eds., 1997, Springer Verlag).

Definitions

“Connective tissue growth factor” or “CTGF” refers to the amino acidsequences of substantially purified CTGF derived from any species,particularly a mammalian species, including rat, rabbit, bovine, ovine,porcine, murine, equine, and hominid, preferably the human species, andfrom any source, whether natural, synthetic, semi-synthetic, orrecombinant.

The term “N-terminal fragment” of CTGF refers to any polypeptidecomprising sequences derived from the amino-terminal portion of a CTGFpolypeptide, or to any variants, or fragments thereof. N-terminalfragments can include all, none, or portions of CTGF from the initialmethionine residue through the cysteine-free “hinge” region as shown inFIGS. 1A and 1B. Further, N-terminal fragments can include all, none, orportions of the insulin growth factor-binding protein motif and/or thevon Willebrand type C domain (SEQ ID NO:21) as shown in FIG. 1B.N-terminal fragments of CTGF can also include all, none, or portions ofthe cysteine-free region. Further, N-terminal fragments of CTGF can beany fifteen or more contiguous amino acids contained within anypreceding N-terminal fragment defined above.

In one aspect, “N-terminal fragment” of CTGF refers to polypeptidesequences derived from the amino-terminal portion of human CTGF. Suchfragments can encompass the entire region from amino acid residue 1 toabout amino acid residue 198 of SEQ ID NO:2, or from about amino acid 23to about amino acid 198 of SEQ ID NO:2. The boundary of the N-terminalfragment within the hinge region may be optionally defined by one ofseveral protease cleavage sites defined in SEQ ID NO:2, such aschymotrypsin cleavage sites between residues 179 and 180, betweenresidues 182 and 183, and between residues 188 and 189; plasmin cleavagesites between residues 183 and 184, and between residues 196 and 197;and a bone morphogenetic protein-1 cleavage site between residues 169and 170. Additionally, N-terminal fragments of human CTGF can includeall, none, or portions of the region from amino acid 27 to amino acid 97of SEQ ID NO:2, amino acid 103 to amino acid 166 of SEQ ID NO:2, oramino acid 167 to amino acid 198 of SEQ ID NO:2. Further, N-terminalfragments of human CTGF can be any fifteen or more contiguous aminoacids contained within any preceding N-terminal fragment defined above.

In specific embodiments, the CTGF N-terminal fragments of the presentinvention comprise sequences selected from the following regions ofhuman CTGF (SEQ ID NO:2) and orthologous fragments thereof derived froma different species, particularly a mammalian species including rat,rabbit, bovine, ovine, porcine, murine, and equine: amino acid residue23 to amino acid residue 96 (encoded by exon 2); amino acid residue 27to amino acid residue 97 (IGF-BP motif); amino acid residue 97 to aminoacid residue 180 (encoded by exon 3); amino acid residue 103 to aminoacid residue 166 (VWC domain); amino acid residue 167 to amino acidresidue 198 (cysteine-free hinge); amino acid residue 23 to amino acidresidue 180 (encoded by exons 2 and 3); amino acid residue 27 to aminoacid residue 166 (IGF-BP and VWC); and amino acid residue 23 to aminoacid residue 198. (See FIG. 1B.)

The term “C-terminal fragment” of CTGF refers to any polypeptidecomprising sequences derived from the carboxy-terminal portion of a CTGFamino acid polypeptide sequence, or to any variants, or fragmentsthereof. C-terminal fragments of CTGF can include all, none, or portionsof the cysteine-free region of CTGF polypeptide (amino acid 167 to aminoacid 198 of SEQ ID NO:2).

The C-terminal fragments can include all, none, or portions of CTGF fromthe cysteine-free hinge region to the end of the protein. Further,C-terminal fragments can include all, none, or portions of thethrombospondin motif and/or the cysteine-knot motif. Further, C-terminalfragments of CTGF can be any fifteen or more contiguous amino acidscontained within any preceding C-terminal fragment defined above.

In some aspects, C-terminal fragments can encompass the entire regionfrom amino acid residue 181 to about amino acid residue 349 of SEQ IDNO:2. The boundary of the C-terminal fragment within the hinge regionmay be optionally defined by one of several protease cleavage sitesdefined in SEQ ID NO:2, such as chymotrypsin, plasmin, and bonemorphogenetic protein-1 cleavage sites defined above. Additionally,C-terminal fragments comprise sequences selected from the followingregions of human CTGF (SEQ ID NO:2) and orthologous fragments thereofderived from a different species, particularly a mammalian speciesincluding rat, rabbit, bovine, ovine, porcine, murine, and equine: aminoacid 201 to amino acid 242 of SEQ ID NO:2, amino acid 247 to amino acid349 of SEQ ID NO:2, amino acid 248 to amino acid 349 of SEQ ID NO:2, oramino acid 249 to amino acid 346 of SEQ ID NO:2. Further, C-terminalfragments of human CTGF can be any fifteen or more contiguous aminoacids contained within any preceding C-terminal fragment defined above.

The terms “cysteine-free region” or “hinge region” of CTGF refer to anypolypeptide derived from about amino acid residue 167 to about aminoacid residue 198 of human CTGF (SEQ ID NO:2) and orthologous fragmentsthereof derived from a different species, particularly a mammalianspecies including rat, rabbit, bovine, ovine, porcine, murine, andequine.

The terms “amino acid sequence” or “polypeptide” or “polypeptides” asused herein refer to oligopeptide, peptide, polypeptide, or proteinsequences, and fragments thereof, and to naturally occurring orsynthetic molecules. A polypeptide or amino acid fragment is any portionof a polypeptide that retains at least one structural and/or functionalcharacteristic of the polypeptide. CTGF fragments include any portion ofa CTGF polypeptide sequence that retains at least one structural orfunctional characteristic of CTGF. Where “amino acid sequence” isrecited to refer to the polypeptide sequence of a naturally occurringprotein molecule, “amino acid sequence” and like terms are not meant tolimit the amino acid sequence to the complete native sequence associatedwith the recited protein molecule.

The term “immunogenicity” relates to the ability of a substance, whenintroduced into the body, to stimulate the immune response and theproduction of an antibody. An agent displaying the property ofimmunogenicity is referred to as being immunogenic. Immunogenic agentscan include, but are not limited to, a variety of macromolecules suchas, for example, proteins, lipoproteins, polysaccharides, nucleic acids,bacteria and bacterial components, and viruses and viral components.Immunogenic agents often have a molecular weight greater than 10 kDa.Antigenic fragments refer to fragments of CTGF polypeptide, preferably,fragments of about five to fifteen amino acids in length, that retain atleast one biological or immunological aspect of CTGF polypeptideactivity.

The term “antibody” refers to intact molecules as well as to fragmentsthereof, such as Fab, F(ab′)2, and Fv fragments, which are capable ofbinding the epitopic determinant, and include polyclonal and monoclonalantibodies. Antibodies that bind CTGF or fragments of CTGF can beprepared using intact polypeptides or using fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide oroligopeptide used to immunize an animal (e.g., a mouse, rat, rabbit,chicken, turkey, goat, etc.) can be derived from the translation of RNA,or synthesized chemically, and can be conjugated to a carrier protein ifdesired. Commonly used carriers chemically coupled to peptides include,for example, bovine serum albumin, thyroglobulin, and keyhole limpethemocyanin (KLH).

The term “monoclonal antibody” as used herein refers to a substantiallyhomogeneous population of antibodies, i.e., the individual antibodiescomprising the population are identical in specificity and affinityexcept for possible naturally occurring mutations that may be present inminor amounts. Note that a monoclonal antibody composition may containmore than one monoclonal antibody.

The monoclonal antibodies included within the scope of the inventioninclude hybrid and recombinant antibodies (e.g., “humanized” antibodies)regardless of species of origin or immunoglobulin class or subclassdesignation, as well as antibody fragments (e.g., Fab, F(ab′)2, and Fv),having at least one of the distinct characteristics of the antibodiesdescribed herein. Preferred embodiments include antibodies capable ofbinding to substantially the same epitope as that recognized bymonoclonal antibody mAb1 and/or have affinity for that epitope that isgreater than or equal to the affinity of mAb1.

The term “monoclonal” indicates the character of the antibody as asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies of the invention may bemade using the hybridoma method first described by Kohler and Milstein(1975, Nature 256:495-497), or may be made by recombinant DNA methods.For example, see Celltech Therapeutics Ltd., European Patent No. EP-0120 694; Cabilly et al., U.S. Pat. No. 4,816,567; or Mage and Lamoyi(1987; In: Monoclonal Antibody Production Techniques and Applications,Marcel Dekker, Inc., New York, pp. 79-97).

The term “neutralizing antibody” as used herein refers to an antibody,preferably a monoclonal antibody, that is capable of substantiallyinhibiting or eliminating a biological activity of CTGF. Typically, aneutralizing antibody will inhibit binding of CTGF to a cofactor such asTGFβ, to a CTGF-specific receptor associated with a target cell, or toanother biological target. In a particular embodiment, a neutralizingantibody will inhibit a biological activity of CTGF to a degreeapproximately equal to or greater than mAb1. Preferably, a neutralizingantibody will inhibit a biological activity of CTGF to a degreeapproximately equal to or greater than CLN1.

The phrase “CTGF-associated disorders” as used herein refers toconditions and diseases associated with abnormal or altered expressionor activity of CTGF. Abnormal expression of CTGF has been associatedwith cell proliferative disorders, such as those caused by endothelialcell proliferation; cell migration; tumor-like growths; general tissuescarring; and various diseases characterized by inappropriate depositionof extracellular matrix.

CTGF-associated disorders include, but are not limited to, disordersinvolving angiogenesis and other processes which play a central role inconditions such as proliferative vitreoretinopathy; cancer, includingacute lymphoblastic leukemia, dermatofibromas, breast cancer, breastcarcinoma, glioma and glioblastoma, rhabdomyosarcoma and fibrosarcoma,desmoplasia, angiolipoma, angioleiomyoma, desmoplastic cancers, andprostate, ovarian, colorectal, pancreatic, gastrointestinal, and livercancer; other tumor growth and metastases; etc.

CTGF-associated disorders also include fibrotic disorders and relatedconditions, such as excessive scarring resulting from localized orsystemic fibrosis, chronic or acute fibrosis of organs such as thekidney, lungs, liver, eyes, heart, skin, etc.; or a tissue selectedfrom, but not limited to, epithelial, endothelial, and connectivetissue. Fibrosis can also occur in the eye and joints. SuchCTGF-associated disorders include, for example, cardiac fibrosis,including cardiac reactive fibrosis or cardiac remodeling followingmyocardial infarction or congestive heart failure; pulmonary disorders,including interstitial pulmonary fibrosis, etc.; fibrosis associatedwith dialysis including peritoneal dialysis, e.g., continuous ambulatoryperitoneal dialysis (CAPD); peridural fibrosis; kidney fibrosis;pulmonary fibrosis; interstitial fibrosis; skin fibrosis; and fibrosisresulting from acute or repetitive traumas, including surgery,chemotherapy, radiation treatment, allograft rejection, chronic andacute transplant rejection (e.g., kidney, liver, or other organ);bronchiolitis obliterans, e.g., following lung transplant; andinflammation and infection, e.g., due to disease or injury.

Additionally, CTGF-associated disorders include, but are not limited to,sclerotic conditions, including systemic sclerosis, scleroderma,keloids, hypertrophic scarring, and other dermatological diseases andconditions; atherosclerosis, such as conditions involvingatherosclerotic plaques and atherosclerosis associated with diabetes,peritoneal dialysis, etc.; arthritis, including rheumatoid arthritis,osteoarthritis, and other joint inflammatory conditions, etc.;interstitial diseases, including interstitial fibrosis; Crohn's disease;inflammatory bowel disease; retinopathies, including, for example,proliferative vitreoretinopathy, non-proliferative diabetic retinopathy,proliferative diabetic retinopathy, and macular degeneration (includingage-related and juvenile (Stargardt's) disease, and pigment epithelialdetachment); nephropathies, including diabetic nephropathy,IgA-associated nephropathy, nephropathy due to toxicity, lupus kidneydisease, etc.; and conditions associated with chemical toxicity tubuledestruction.

CTGF-associated disorders also include, but are not limited to,disorders due to hyperglycemia, hypertension, advanced glycationendproduct (AGE) formation, etc. Such disorders may occur, e.g., due todiabetes, obesity, etc., and include diabetic nephropathy, retinopathy,and cardiovascular disease. Further, CTGF-associated disorders may becaused by any initiating factor including, but not limited to, exposureto chemicals or biological agents, inflammatory response, autoimmunereaction, trauma, surgical procedures, etc. In some embodiments, themethods are used to treat a patient predisposed to a CTGF-associateddisorder due to a condition including, but not limited to, myocardialinfarction, arthritis, and local or systemic inflammation.

The “proliferative” processes and disorders referred to herein includepathological states characterized by the continual multiplication ofcells resulting in an overgrowth of a cell population within a tissue.The cell populations are not necessarily transformed, tumorigenic ormalignant cells, but can include normal cells as well. For example, CTGFmay be involved pathologically by inducing a proliferative lesion in theintimal layer of an arterial wall, resulting in atherosclerosis, or bystimulating neovascularization.

“Cancer” refers to any autonomous growth of tissue, includinguncontrolled, abnormal growth of cells, or to any malignant tumor ofpotentially unlimited growth that expands locally by invasion andsystemically by metastasis. Cancer also refers to any abnormal statemarked by a cancer.

The term “fibrosis” refers to abnormal processing of fibrous tissue, orfibroid or fibrous degeneration. Fibrosis can result from variousinjuries or diseases, and can often result from chronic transplantrejection relating to the transplantation of various organs. Fibrosistypically involves the abnormal production, accumulation, or depositionof extracellular matrix components, including overproduction andincreased deposition of, for example, collagen and fibronectin.“Fibrosis” is used herein in its broadest sense referring to any excessproduction or deposition of extracellular matrix proteins. There arenumerous examples of fibrosis, including the formation of scar tissuefollowing a heart attack, which impairs the ability of the heart topump. Diabetes frequently causes damage/scarring in the kidneys, whichleads to a progressive loss of kidney function; and in the eyes, whichcauses loss of vision. After surgery, scar tissue can form betweeninternal organs causing contracture, pain, and in some cases,infertility. Major organs such as the heart, kidney, liver, eye, andskin are prone to chronic scarring, commonly associated with otherdiseases. Hypertrophic scars (non-malignant tissue bulk) are a commonform of fibrosis caused by burns and other trauma. In addition, thereare a number of other fibroproliferative disorders, includingscleroderma, keloids, and atherosclerosis, which are associatedrespectively with general tissue scarring, tumor-like growths in theskin, or sustained scarring of blood vessels which impairs bloodcarrying ability.

The terms “nucleic acid” or “polynucleotide” or “polynucleotides” referto oligonucleotides, nucleotide sequences, or polynucleotides, or anyfragments thereof, and to DNA or RNA of natural or synthetic originwhich may be single- or double-stranded and may represent the sense orantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material, natural or synthetic in origin. Polynucleotidefragments are any portion of a polynucleotide sequence that retains atleast one structural or functional characteristic of the polynucleotide.Polynucleotide fragments can be of variable length, for example, greaterthan 60 nucleotides in length, at least 100 nucleotides in length, atleast 1000 nucleotides in length, or at least 10,000 nucleotides inlength.

“Altered” polynucleotides include those with deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent polypeptide. Includedwithin this definition are sequences displaying polymorphisms that mayor may not be readily detectable using particular oligonucleotide probesor through deletion of improper or unexpected hybridization to alleles,with a locus other than the normal chromosomal locus for the subjectpolynucleotide sequence.

“Altered” polypeptides may contain deletions, insertions, orsubstitutions of amino acid residues, which produce a silent change andresult in a functionally equivalent polypeptide. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the biological orimmunological activity of the encoded polypeptide is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

A polypeptide or amino acid “variant” is an amino acid sequence that isaltered by one or more amino acids from a particular amino acidsequence. A polypeptide variant may have conservative changes, wherein asubstituted amino acid has similar structural or chemical properties tothe amino acid replaced, e.g., replacement of leucine with isoleucine. Avariant may also have non-conservative changes, in which the substitutedamino acid has physical properties different from those of the replacedamino acid, e.g., replacement of a glycine with a tryptophan. Analogousminor variations may also include amino acid deletions or insertions, orboth. Preferably, amino acid variants retain certain structural orfunctional characteristics of a particular polypeptide. Guidance indetermining which amino acid residues may be substituted, inserted, ordeleted may be found, for example, using computer programs well known inthe art, such as LASERGENE software (DNASTAR Inc., Madison, Wis.).

A polynucleotide variant is a variant of a particular polynucleotidesequence that preferably has at least about 80%, more preferably atleast about 90%, and most preferably at least about 95% polynucleotidesequence similarity to the particular polynucleotide sequence. It willbe appreciated by those skilled in the art that as a result of thedegeneracy of the genetic code, a multitude of variant polynucleotidesequences encoding a particular protein, some bearing minimal homologyto the polynucleotide sequences of any known and naturally occurringgene, may be produced. Thus, the invention contemplates each and everypossible variation of polynucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard codon tripletgenetic code, and all such variations are to be considered as beingspecifically disclosed.

A “deletion” is a change in an amino acid or nucleotide sequence thatresults in the absence of one or more amino acid residues ornucleotides.

The terms “insertion” or “addition” refer to a change in a polypeptideor polynucleotide sequence resulting in the addition of one or moreamino acid residues or nucleotides, respectively, as compared to thenaturally occurring molecule.

The term “functional equivalent” as it is used herein refers to apolypeptide or polynucleotide that possesses at least one functionaland/or structural characteristic of a particular polypeptide orpolynucleotide. A functional equivalent may contain modifications thatenable the performance of a specific function. The term “functionalequivalent” is intended to include fragments, mutants, hybrids,variants, analogs, or chemical derivatives of a molecule.

The term “microarray” refers to any arrangement of nucleic acids, aminoacids, antibodies, etc., on a substrate. The substrate can be anysuitable support, e.g., beads, glass, paper, nitrocellulose, nylon, orany appropriate membrane, etc. A substrate can be any rigid orsemi-rigid support including, but not limited to, membranes, filters,wafers, chips, slides, fibers, beads, including magnetic or nonmagneticbeads, gels, tubing, plates, polymers, microparticles, capillaries, etc.The substrate can provide a surface for coating and/or can have avariety of surface forms, such as wells, pins, trenches, channels, andpores, to which the nucleic acids, amino acids, etc., may be bound.

The term “sample” is used herein in its broadest sense. Samples may bederived from any source, for example, from bodily fluids, secretions,tissues, cells, or cells in culture including, but not limited to,saliva, blood, urine, serum, plasma, vitreous, synovial fluid, cerebralspinal fluid, amniotic fluid, and organ tissue (e.g., biopsied tissue);from chromosomes, organelles, or other membranes isolated from a cell;from genomic DNA, cDNA, RNA, mRNA, etc.; and from cleared cells ortissues, or blots or imprints from such cells or tissues. Samples may bederived from any source, such as, for example, a human subject, or anon-human mammalian subject, etc. Also contemplated are samples derivedfrom any animal model of disease. A sample can be in solution or can be,for example, fixed or bound to a substrate. A sample can refer to anymaterial suitable for testing for the presence of CTGF or of fragmentsof CTGF or suitable for screening for molecules that bind to CTGF or tofragments thereof. Methods for obtaining such samples are within thelevel of skill in the art.

The term “hybridization” refers to the process by which a nucleic acidsequence binds to a complementary sequence through base pairing.Hybridization conditions can be defined by, for example, theconcentrations of salt or formamide in the prehybridization andhybridization solutions, or by the hybridization temperature, and arewell known in the art. Hybridization can occur under conditions ofvarious stringency.

In particular, stringency can be increased by reducing the concentrationof salt, increasing the concentration of formamide, or raising thehybridization temperature. For example, for purposes of the presentinvention, hybridization under high stringency conditions might occur inabout 50% formamide at about 37° C. to 42° C., and under reducedstringency conditions in about 35% to 25% formamide at about 30° C. to35° C. In particular, hybridization generally occurs in conditions ofhighest stringency at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS, and 200μg/ml sheared and denatured salmon sperm DNA.

The temperature range corresponding to a particular level of stringencycan be further narrowed by methods known in the art, for example, bycalculating the purine to pyrimidine ratio of the nucleic acid ofinterest and adjusting the temperature accordingly. To removenonspecific signals, blots can be sequentially washed, for example, atroom temperature or up to and including 60° C., under increasinglystringent conditions of up to 0.1×SSC and 0.5% SDS. Variations on theabove ranges and conditions are well known in the art.

Invention

The present invention provides antibodies that specifically bind toConnective Tissue Growth Factor (CTGF). The antibodies are polyclonal ormonoclonal antibodies, preferably monoclonal antibodies, and morepreferably human monoclonal antibodies. The antibodies are directedtoward the N-terminal fragment of CTGF, shown in FIG. 1. Morespecifically, the antibodies are directed toward a fragment of CTGFextending from about residue 97 to about residue 180 of SEQ ID NO:2. Inparticular embodiments, the antibodies are directed toward a fragment ofCTGF extending from about residue 103 to about residue 164, and moreparticularly a fragment from about residue 134 to about residue 158 ofSEQ ID NO:2. More specifically, the antibodies are directed toward afragment of CTGF extending from about residue 143 to about residue 154of SEQ ID NO:2.

In particular embodiments, the antibodies neutralize a biologicalactivity of CTGF. Biological activities of CTGF include cellproliferation, differentiation, gene expression, etc. In particularembodiments, the biological activity is selected from the groupconsisting of cellular differentiation, e.g., differentiation ortransdifferentiation of fibroblasts, myofibroblasts, endothelial cells,etc., from various precursor cells; induction of expression of proteinsinvolved in extracellular matrix formation and remodeling including,e.g., type I collagen, fibronectin, etc.; cooperative induction ofsignaling cascades associated with various factors including, but notlimited to, TGF-β, IGF, VEGF, angiotensin II, endothelin, etc.; andcellular response to various environmental stimuli including, but notlimited to, increased glucose (hyperglycemia), increased mechanicalstress (hypertension), etc.

Although the invention is not to be limited by the mechanism by whichthe antibodies neutralize CTGF activity, the antibodies may bind to andprevent CTGF from interacting with specific cell receptors. Thereceptors may have high binding affinity for CTGF and, by binding toCTGF, stimulate an intracellular signal that leads to proliferation,differentiation, induction of gene expression, and/or change in cellularmorphology or function. The particular biological response of a cell toCTGF depends on the cell and the current state of the surroundingmilieu. Alternatively, the receptors may have low binding affinity forCTGF and, by binding to CTGF, may, e.g., position CTGF relative to highaffinity receptors to facilitate recognition for and response to CTGF.Alternatively, the antibodies may bind CTGF within tissues or organs andfacilitate titration or elimination of CTGF from the body.

Alternatively or in conjunction with the mechanisms described above, theantibodies may bind to and prevent CTGF from interacting with secretedor membrane-bound cofactors. Such cofactors specifically include membersof the TGFβ superfamily including, e.g., TGFβ-1, -2, and -3; activin-A,-B, -C, and -E; BMP-2, -3, -4, -5, -6, -7, -8a, -8b, -9, -10, -11, and-15; and GDF-3, -5, -6, -7, -9, and -10. For example, CTGF has beenshown to bind to TGFβ-1 and BMP-4 and modulate their activity. (Abreu etal. (2002) Nat Cell Biol 4: 599-604.) The present invention providesevidence that the region of CTGF that binds to TGFβ is encoded by exon 3(FIG. 1B; nucleotide 418 to nucleotide 669 of SEQ ID NO:1) andantibodies that bind within this region prevent interaction between CTGFand TGFβ. (Example 12, infra.) Further, antibodies that bind within thisregion of CTGF have been shown to neutralize specific CTGF-associatedprocesses in animal models. For example, antibodies that bind withinthis region of CTGF have been shown to specifically inhibit cellmigration in ex vivo assays, and reduce fibrosis in animal models.Exemplary antibodies of the invention are mAb1 and CLN1; antibody CLN1is produced by the cell line defined by ATCC Accession No. PTA-6006,deposited with the American Type Culture Collection (ATCC, 10801University Boulevard, Manassas Va. 20110-2209) on 20 May 2004. TheChinese Hamster Ovary (CHO) cell line identified as ATCC Accession No.PTA-6006, which produces the human monoclonal antibody CLN-1, wasdeposited with the ATCC under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures at ATCC for atleast 30 years from the date of deposit, or for the enforceable life ofthe patent, or for a period of 5 years after the date of the most recentrequest for furnishing a sample of the deposited material, whichever islongest. The organisms will be made available by ATCC under the terms ofthe Budapest Treaty and the cell line will be irrevocably and withoutrestriction or condition released to the public upon issuance of apatent. Also, if the culture deposit should die or be lost or destroyedwhen cultivated under suitable conditions, it will be promptly replacedon notification with a viable specimen of the same culture. Availabilityof a deposited strain is not to be construed as a license to practicethe invention in contravention of the rights granted under the authorityof any government in accordance with its patent laws.

Regardless of the mechanism of action, the present invention providesmethods of using the antibodies to treat various diseases and disordersassociated with CTGF. Diseases and disorders associated with CTGFinclude, but are not limited to, nephropathies, pulmonary fibroses,retinopathies, scleroderma, liver fibroses, heart failure, arthritis,and atherosclerosis. Additionally, disorders associated with CTGF occurdue to various factors including, but not limited to, hyperglycemia,hypertension, diabetes, obesity, etc; and include diabetic nephropathy,retinopathy, cardiovascular disease, and the like. As CTGF isoverexpressed in a wide variety of diseases including those listedabove, the invention contemplates treating patients having aCTGF-associated disorder with a CTGF antibody to improve or stabilizethe pathology, retain or restore organ function, improve the quality oflife, and prolong survival.

For example, the antibodies are particularly directed to regions of CTGFinvolved in biological activities associated with both fibrotic andnon-fibrotic aspects of various disorders including, e.g., interstitialpulmonary fibrosis, diabetic nephropathy and retinopathy, maculardegeneration, etc. The invention also relates to methods of using theantibodies to treat disorders associated with CTGF including localizedand systemic fibrotic disorders, such as those of the lung, liver,heart, skin, and kidney, etc.; and localized scar formation due to,e.g., trauma, surgical procedures, etc.

The antibodies of the invention can also be used in any method thatinvolves binding to CTGF. Such methods include purification of CTGF orfragments of CTGF, e.g., by affinity chromatography; detection of CTGFor fragments of CTGF in a sample, e.g., using ELISA orimmunohistochemical techniques; diagnosing a CTGF-associated disorder byusing the method of detecting CTGF to measure CTGF levels in a patientsample and comparing the level of CTGF in the sample to a standard.

Antibodies Directed to CTGF

Modulation of the amount and/or activity of secreted cellular factorsusing, e.g., monoclonal antibodies, has been demonstrated, and severaltherapeutic antibodies have been approved or are under development.(See, e.g., Abciximab (Reopro; Centocor, Inc., Malvern Pa.), Infliximab(Remicade; Maini et al. (1998) Arthritis Rheum 41:1552-1563; Targan etal. (1997) N Engl J Med 337:1029-1035); Basiliximab (Simulect) andDaclizumab (Zenapax) (Bumgardner et al. (2001) Transplantation72:839-845; Kovarik et al. (1999) Transplantation 68:1288-1294); andTrastuzumab (Herceptin; Baselga (2001) Ann Oncol 12 Suppl 1:S49-55.))Numerous methods of producing antibodies, including production inanimals, plants, fungi, and bacteria; synthetic construction; and exvivo culture; are known and available to those of skill in the art.

The antibodies of the invention may be prepared using any technique thatprovides for the production of antibody molecules. Techniques for invivo and in vitro production of either monoclonal or polyclonalantibodies are well known in the art. (See, e.g., Pound (1998)Immunochemical Protocols, Humana Press, Totowa N.J.; Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York; Goding (1986) Monoclonal Antibodies: Principles and Practice,2^(nd) Edition, Academic Press; Schook (1987) Monoclonal AntibodyProduction Techniques and Applications, Marcel Dekker, Inc.) Theproduction of chimeric antibodies is also well known in the art, as isthe production of single-chain antibodies. (See, e.g., Morrison et al.(1984) Proc Natl Acad Sci USA 81:6851-6855; Neuberger et al. (1984)Nature 312:604-608; Takeda et al. (1985) Nature 314:452-454.) Antibodieswith related specificity, but of distinct idiotypic composition, may begenerated by a variety of available means, for example, by chainshuffling from random combinatorial immunoglobin libraries. (See, e.g.,Burton (1991) Proc Natl Acad Sci USA 88:11120-11123.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents. (See, e.g., Orlandi et al. (1989)Proc Natl Acad Sci USA 86:3833-3837; Winter and Milstein (1991) Nature349:293-299.) Antibody fragments that contain specific binding sites forthe target polypeptide may also be generated. Such antibody fragmentsinclude, but are not limited to, F(ab′)2 fragments, which can beproduced by pepsin digestion of the antibody molecule, and Fabfragments, which can be generated by reducing the disulfide bridges ofthe F(ab′)2 fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity. (See, e.g., Huse et al. (1989)Science 254:1275-1281.)

Monoclonal antibodies of the invention may also be prepared using thehybridoma method (see, e.g., Kohler and Milstein (1975) Nature256:495-497) or by recombinant DNA methods (see, e.g., CelltechTherapeutics Ltd., European Patent No. EP 0 120 694; Cabilly et al.,U.S. Pat. No. 4,816,567; and Mage and Lamoyi (1987) In: MonoclonalAntibody Production Techniques and Applications, Marcel Dekker, Inc.,New York, pp. 79-97).

In the hybridoma method, a mouse or other appropriate host animal isimmunized with CTGF or a fragment thereof by subcutaneous,intraperitoneal, or intramuscular routes to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the polypeptide used for immunization. Alternatively, the hostanimal may be a transgenic mammal having transgenes encoding humanimmunoglobulin genes and having inactivated endogenous immmunoglobulinloci. The transgenic mammal responds to immunogens by producing humanantibodies. (See, e.g., Lonberg et al., WO 93/12227 (1993), U.S. Pat.No. 5,877,397, and Nature 148:1547-1553 (1994); and Kucherlapati et al.(1991) WO 91/10741.) Alternatively, lymphocytes may be immunized invitro and then fused with myeloma cells using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell. (See, e.g.,Goding (1986) Monoclonal Antibodies: Principles and Practice, 2^(nd)Edition, Academic Press, pp. 59-103.) Alternatively, human somatic cellscapable of producing antibody, specifically B lymphocytes, are suitablefor fusion with myeloma cell lines. While B lymphocytes from biopsiedspleens, tonsils or lymph nodes of an individual may be used, the moreeasily accessible peripheral blood B lymphocytes are preferred. Inaddition, human B cells may be directly immortalized by the Epstein-Barrvirus. (See, e.g., Cole et al. (1995) Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96.)

Preferred myeloma cell lines for use in hybridoma-producing fusionprocedures are those that fuse efficiently, support stable high-levelexpression of antibody by the selected antibody-producing cell, haveenzyme deficiencies that render them incapable of growing in certainselective media which support the growth of the desired hybridomas, andthat do not themselves produce antibody. Examples of myeloma cell linesthat may be used for the production of hybridomas in the presentinvention include P3X63Ag8, P3X63Ag8-653, NS1/1.Ag 4.1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7, S194/5XX0 Bul, all derived from mice;R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210, all derived from rats; andU-266, GM1500-GRG2, LICR-LON-HMy2, UC729-6, all derived from humans.(See, e.g., Goding (1986) Monoclonal Antibodies: Principles andPractice, 2^(nd) Edition, Academic Press, pp. 65-66; and Campbell (1984)In: Monoclonal Antibody Technology: Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 13 (Burden and Von Knippenberg,eds.) Amsterdam, Elseview, pp. 75-83.)

The hybridoma cells are seeded and grown in a suitable culture mediumthat preferably contains one or more substances that inhibit the growthor survival of the unfused, parental myeloma cells. For example, if theparental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include a substance such as hypoxanthine,aminopterin, and thymidine (HAT medium) that prevents the growth ofHGPRT-deficient cells.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against CTGF or fragmentsof CTGF. Preferably, the binding specificity is determined by affinitychromatography, immunoprecipitation or by an in vitro binding assay,such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay(ELISA), or by fluorescence-activated cell sorting (FACS) analysis. Themonoclonal antibodies of the invention are those that bind to CTGF, andadditionally those that neutralize a CTGF biological activity, asexemplified infra.

The antibodies produced, e.g., as described supra, are optionallyscreened to detect antibodies that bind to substantially the N-terminalfragment of CTGF. In one embodiment, the antibodies are directed towarda fragment of CTGF extending from about residue 24 to about residue 180of SEQ ID NO:1. In another embodiment, the antibodies are directedtoward a fragment of CTGF extending from about residue 96 to aboutresidue 180 of SEQ ID NO:1. In a particular embodiment, the screendetects antibodies that bind to substantially the same epitoperecognized by antibody mAb1 as determined, e.g., by competition assaysof the sort described infra. In another particular embodiment, thescreen detects antibodies that bind to substantially the same epitoperecognized by antibody CLN1 as determined, e.g., by competition assaysof the sort described infra. It should be kept in mind that “sameepitope” does not mean the exact amino acid or carbohydrate to which thebenchmark antibody binds, as may be determined, for example, by epitopemapping using alanine scanned variants of CTGF. “Same epitope” means theCTGF domain that is blocked by the binding to CTGF of the nativebenchmark antibody in intact form. Of course, “same epitope” includesthe CTGF domain residues or carbohydrate that structurally interacts orbinds to the benchmark complementarity determining regions (CDRs) ofmAb1 or CLN1.

In a preferred embodiment of the invention, the monoclonal antibody willhave an affinity that is equal to or greater than that of mAb1, asdetermined, for example, by the Scatchard analysis of Munson and Pollard(1980, Anal Biochem 107:220).

After hybridoma cells are identified that produce neutralizingantibodies of the desired specificity and affinity, the clones typicallyare subcloned by limiting dilution procedures and grown by standardmethods. (Goding (1986) Monoclonal Antibodies: Principles and Practice,2^(nd) Edition, Academic Press, pp. 59-104.) Suitable culture media forthis purpose include, for example, Dulbecco's Modified Eagle's Medium orRPMI-1640 medium. In addition, the hybridoma cells may be grown in vivoas ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures, e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the antibody heavy and light chains. Once isolated, the DNA canbe ligated into expression or cloning vectors, which are thentransfected into host cells such as simian COS cells, Chinese Hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein. The cells so transformed are cultured underconditions suitable for the synthesis of monoclonal antibodies in therecombinant host cell culture. An exemplary cell line is defined by ATCCAccession No. PTA-6006, deposited with the ATCC on 20 May 2004.

The DNA optionally is modified in order to change the character of theencoded immunoglobulin. Variants of immunoglobulins are well known. Forexample, chimeric antibodies are made by substituting the codingsequence for heavy and light chain constant domains from one species,e.g., mouse, with the homologous sequences from another species, e.g.,human. (See, e.g., Boss et al., International Publication No. WO84/03712; Cabilly et al., U.S. Pat. No. 4,816,567; or Morrison et al.(1984) Proc Nat Acad Sci 81:6851.) In a particular embodiment, humanizedforms of murine antibodies can be made by substituting thecomplementarity determining regions (CDRs), i.e., variable domains, of amouse antibody into a framework domain, i.e., constant region, of ahuman antibody. (See, e.g., International Publication No. WO 92/22653.)In some embodiments, selected murine framework residues also aresubstituted into the human recipient immunoglobulin. In addition, the Fcdomain chosen can be any of IgA, IgD, IgE, IgG-1, IgG-2, IgG-3, IgG-4,or IgM. The Fc domain optionally is capable of effector functions suchas complement binding.

Anti-CTGF antibodies of the present invention may also be fused tomoieties that provide additional capabilities, such as detection orcytotoxic effects. Fusions of the immunoglobulins of this invention andcytotoxic moieties are made, for example, by ligating to theimmunoglobulin coding sequence all or part of the coding sequence for acytotoxic non-immunoglobulin polypeptide. Such non-immunoglobulinpolypeptides include polypeptide toxins such as ricin, diphtheria toxin,or Pseudomonas exotoxin. The conjugates can also be prepared by in vitromethods. For example, immunotoxins may be constructed using a disulfideexchange reaction or by forming a thioether bond between theimmunoglobulin and the toxin polypeptide. Examples of suitable reagentsfor this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate. Typically such non-immunoglobulin fusionpolypeptides are substituted for the constant domains of an antibody ofthe invention. Alternatively, they are substituted for the variabledomains of one antigen-combining site of an antibody of the invention.

Substitution of the Fv or CDRs of an antibody having specificity for anon-CTGF antigen will create a chimeric antibody comprising oneantigen-combining site having specificity for CTGF and anotherantigen-combining site having specificity for a different antigen. Insuch embodiments, the light chain is deleted and the Fv of the heavychain is substituted with the desired polypeptide. These antibodies aretermed bivalent or polyvalent, depending upon the number ofimmunoglobulin “arms” possessed by the Fc domain employed; for example,IgGs will be bivalent and IgMs will be polyvalent. Aside from thenonimmunoglobulins mentioned above, the antibody also is renderedmultivalent by recombination of antibodies that have more than onespecificity. For instance, the antibody in some embodiments is capableof binding CTGF as described elsewhere herein, but is also capable ofbinding a second growth factor, e.g., TGFβ, VEGF, FGF, other CCN familymembers, e.g., CYR61, and the like, or a cytokine. Exemplary antibodiesdirected against these factors are well-known. The multispecific,multivalent antibodies are made by cotransforming a cell with DNAencoding the heavy and light chains of both antibodies and theproportion of expressed antibodies having the desired structurerecovered by immunoaffinity chromatography or the like. Alternatively,such antibodies are made from monovalent antibodies that are recombinedin vitro in conventional fashion.

Monovalent antibodies also are made by techniques that are conventionalper se. Recombinant expression of light chain and a modified heavy chainis suitable. The heavy chain is truncated generally at any point in theFc region so as to prevent heavy chain crosslinking. Alternatively, therelevant cysteines are substituted with another residue or deleted so asto prevent crosslinking. In vitro methods also are used to producemonovalent antibodies, e.g., Fab fragments are prepared by enzymaticcleavage of intact antibody.

Diagnostics

The antibodies of the present invention can be used to quantitativelyand qualitatively detect CTGF in a sample. Samples can be from anysource, including conditioned media from cells grown in culture; tissuesamples, e.g., tissue biopsies and organ transplants; body fluidsincluding blood, urine, blister fluid, cerebrospinal fluid, vitreous,and synovial fluid; etc. In one embodiment, detection of CTGF is used todiagnose the state of cells grown in culture, e.g., with regard todifferentiation, matrix production, etc. CTGF has various autocrine andparacrine effects on cultured cells, and the level of CTGF associatedwith the cell layer or present in conditioned media may be indicative ofthe current state of the cell or predictive of the future state of thecell. (See, e.g., International Publication No. WO 96/38168.) In otherembodiments, detection of CTGF is used to determine the state of atissue or organ. For example, an organ destined for transplant can beevaluated by measuring CTGF levels, wherein the level of CTGF expressedby cells in the organ indicate the relative health of the organ andsuitability for transplant. CTGF levels can also be determined inbiopsied tissue to determine the status of an organ, or the stage andpotential metastatic potential of a cancer.

In preferred embodiments, the antibodies are used to diagnose a diseaseor disorder associated with CTGF. (See, e.g., International PublicationNo. WO 03/024308.) In one aspect, the invention provides antibodies fordiagnosing a CTGF-associated disorder by obtaining a sample, detectingand quantitating the level of CTGF in the sample, and comparing thelevel of CTGF in the sample to that of a standard amount of CTGF,wherein an increased or decreased amount of CTGF in the sample isindicative of the presence of a CTGF-associated disorder. Disordersassociated with aberrant (e.g., increased or decreased) levels of CTGFinclude, but are not limited to, disorders associated with alteredexpression and deposition of extracellular matrix-associated proteins.Such disorders include, for example, cancers such as breast, pancreatic,and gastrointestinal cancer; atherosclerosis, arthritis, retinopathiessuch as diabetic retinopathy; nephropathies such as diabeticnephropathy; cardiac, pulmonary, liver, and kidney fibrosis, anddiseases associated with chronic inflammation and/or infection.CTGF-associated disorders are also associated with conditions such asmyocardial infarction, diabetes, peritoneal dialysis, chronic and acutetransplant rejection, chemotherapy, radiation therapy, and surgery.

In another aspect, the invention provides antibodies for identifyingwhether or not an individual has a predisposition to develop aCTGF-associated disorder. A predisposition may be initially indicated byhyperglycemia, hypertension, or obesity in a subject. Additionally, apredisposition may be suspected due to an event, e.g., a myocardialinfarction, surgery, orthopedic or paralytic immobilization, congestiveheart failure, pregnancy, or varicosities in the subject.

In another aspect, the invention provides antibodies for monitoring theprogression of a CTGF-associated disorder or monitoring the therapeuticefficacy of treatment of a CTGF-associated disorder. For example, amethod of using the antibodies may comprise obtaining samples from asubject over time; detecting and quantitating the level of CTGF in eachsample; and comparing the level of CTGF in subsequent samples with CTGFlevels in earlier or previous samples. A change in CTGF level betweensamples over time is indicative of the progression of theCTGF-associated disorder or the therapeutic efficacy of treatment of theCTGF-associated disorder.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany moiety capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed. (See, e.g., Hunter et al. (1962)Nature 144:945; David et al. (1974) Biochemistry 13:1014; Pain et al.(1981) J Immunol Meth 40:219; and Nygren (1982) J Histochem Cytochem30:407.) The antibodies of the present invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays. (Zola (1987)In: Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., pp.147-158.)

Competitive binding assays rely on the ability of a labeled standard(which may be CTGF or an immunologically reactive portion thereof) tocompete with the test sample analyte (CTGF) for binding with a limitedamount of antibody. The amount of CTGF in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte that remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insoluble threepart complex. (David and Greene, U.S. Pat. No. 4,376,110.) The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme. An exemplary assay in which theantibodies of the invention may be used, e.g., is described inInternational Publication No. WO 03/024308.

The antibodies of the invention also are useful for in vivo imaging,wherein an antibody labeled with a detectable moiety such as aradio-opaque agent, radioisotope, or fluorescent moiety such as greenfluorescent protein (GFP) is administered to a host, preferably into thebloodstream, and the presence and location of the labeled antibody inthe host is assayed. This imaging technique is useful in the staging andtreatment of CTGF-associated disorders such as fibrotic disorders. Theantibody may be labeled with any moiety that is detectable in a host,whether by nuclear magnetic resonance, radiology, or other detectionmeans known in the art.

Therapeutics

The present invention provides antibodies for treatment of variousdiseases and disorders associated with CTGF. The antibodies of theinvention have been found to reduce the deleterious effects of CTGFproduction or activity in several disorders, as exemplified below.Further, the antibodies show favorable pharmacokinetics making themsuperior therapeutic agents for the treatment of disorders associatedwith CTGF.

The anti-CTGF antibodies of the present invention inhibit development offibrosis in animal models of, e.g., lung and kidney fibrosis.Specifically, the antibodies attenuate bleomycin-induced lung fibrosisin mice by 60-70%, as determined by inhibition of pulmonaryhydroxyproline (collagen) accumulation and histological examination oftissue preparations. Further, the antibodies reduce the accumulation ofcollagen in a rat remnant kidney (i.e., 5/6 nephrectomy) model, and inmice following unilateral ureter obstruction (UUO). The antibodies alsoreduce fibrosis induced by combined subcutaneous or intraperitonealinfusion of CTGF and TGFβ in newborn mice. Additionally, the antibodiesreduce complications associated with organ failure, e.g., improvedkidney function in various models of chronic and acute kidney failure.No toxicity has been observed with these antibodies in animals. As CTGFis overexpressed in a wide variety of fibrotic diseases includingdiffuse and limited scleroderma, osteoarthritis, diabetic nephropathyand retinopathy, etc., the invention contemplates treating patients witha CTGF-associated disorder with a CTGF antibody to improve or stabilizethe pathology, restore organ function, improve the quality of life, andextend survival.

Therefore, the antibodies of the invention are especially useful intherapeutic applications, to prevent or treat CTGF-associated disordersin a subject. Such disorders include, but are not limited to,angiogenesis and other processes which play a central role in conditionssuch as atherosclerosis, glaucoma, etc.; and in cancer, including acutelymphoblastic leukemia, dermatofibromas, breast cancer, breastcarcinoma, glioma and glioblastoma, rhabdomyosarcoma and fibrosarcoma,desmoplasia, angiolipoma, angioleiomyoma, desmoplastic cancers, andprostate, ovarian, colorectal, pancreatic, gastrointestinal, and livercancer and other tumor growth and metastases.

Additionally, the antibodies of the invention are useful in therapeuticapplications to prevent or treat CTGF-associated disorders involvingfibrosis. In one aspect, the antibodies of the invention areadministered to a subject to prevent or treat a CTGF-associated disorderincluding, but are not limited to, disorders exhibiting alteredexpression and deposition of extracellular matrix-associated proteins,e.g., fibrotic disorders. In various aspects, the fibrosis may belocalized to a particular tissue, such as epithelial, endothelial, orconnective tissue; or to an organ, such as kidney, lung, or liver.Fibrosis can also occur in the eye and joints. In other aspects, thefibrosis may be systemic and involve multiple organ and tissue systems.CTGF-associated disorders include, for example, atherosclerosis,arthritis, retinopathies such as diabetic retinopathy; nephropathiessuch as diabetic nephropathy; cardiac, pulmonary, liver, and kidneyfibrosis, and diseases associated with chronic inflammation and/orinfection.

In another aspect, the invention provides antibodies for preventing aCTGF-associated disorder in a subject having a predisposition to developsuch a disorder. A predisposition may include, e.g., hyperglycemia,hypertension, or obesity in the subject. Such disorders may occur, e.g.,due to diabetes, obesity, etc., and include diabetic nephropathy,retinopathy, and cardiovascular disease. Additionally, a predispositionmay be suspected due to an event, e.g., a myocardial infarction,surgery, peritoneal dialysis, chronic and acute transplant rejection,chemotherapy, radiation therapy, trauma, orthopedic or paralyticimmobilization, congestive heart failure, pregnancy, or varicosities inthe subject.

In particular embodiments, as exemplified herein, the antibodies of thepresent invention are administered to a subject to treat fibrosis of anorgan, e.g., lung or kidney. The antibodies are shown herein to providebenefit in various models of lung and kidney fibrosis. (See, e.g.,Examples 7 to 9.) In another particular embodiment, the antibodies ofthe present invention are administered to a subject to reduce local orsystemic sclerosis. (See, e.g., Examples 11 and 12.) In additionalembodiments, the antibodies are administered to a subject to treat orprevent ocular disorders such as proliferative vitreoretinopathy,diabetic retinopathy, macular degeneration, etc. As CTGF is implicatedin a wide variety of disorders, the invention further contemplatestreating patients having a CTGF-associated disorder using an antibody ofthe invention to improve or stabilize pathology and organ function,improve the quality of life, and extend survival.

For therapeutic applications, the antibodies of the invention areadministered to a mammal, preferably a human, in a pharmaceuticallyacceptable dosage form. The antibodies may be administered intravenouslyas a bolus or by continuous infusion over a period of time, and/or byintramuscular, subcutaneous, intra-articular, intrasynovial,intrathecal, intravitreal, intracranial, oral, topical, or inhalationroutes. When the antibody possesses the suitable activity, intratumoral,peritumoral, intralesional, or perilesional routes of administration canalso be utilized to exert local as well as systemic therapeutic effects.

Such dosage forms encompass pharmaceutically acceptable carriers thatare inherently nontoxic and nontherapeutic. Examples of such carriersinclude ion exchangers, alumina, aluminum stearate, lecithin; serumproteins such as human serum albumin; buffers such as phosphate orglycine; sorbic acid, potassium sorbate, partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts; or electrolytes such asprotamine sulfate, sodium chloride, metal salts, colloidal silica,magnesium trisilicate, polyvinyl pyrrolidone, cellulosic polymers, andpolyethylene glycol. Carriers for topical or gel-based forms of antibodyinclude polysaccharides such as sodium carboxymethylcellulose ormethylcellulose, polyvinylpyrrolidone, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol,and wood wax alcohols. Conventional depot forms include, for example,microcapsules, nano-capsules, liposomes, plasters, sublingual tablets,and polymer matrices such as polylactide:polyglycolide copolymers. Whenpresent in an aqueous dosage form, rather than being lyophilized, theantibody typically will be formulated at a concentration of about 0.1mg/ml to 100 mg/ml, although wide variation outside of these ranges ispermitted.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibodiesare administered for preventive or therapeutic purposes, the course ofprevious therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibody issuitably administered to the patient at one time or over a series oftreatments.

Depending on the type and severity of the disease, about 0.015 to 15 mgof antibody/kg of patient weight is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. For repeatedadministrations over several days or longer, depending on the condition,the treatment is repeated until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful and arenot excluded from the present invention.

According to another embodiment of the invention, the effectiveness ofthe antibody in preventing or treating disease may be improved byadministering the antibody serially or in combination with another agentthat is effective for the same clinical objective, such as anotherantibody directed against a different epitope than the principalantibody, or one or more conventional therapeutic agents known for theintended therapeutic indication, e.g. prevention or treatment ofconditions associated with excessive extracellular matrix productionsuch as fibrosis or sclerosis, inhibition of tumor cell growth ormetastasis, inhibition of neovascularization, or reduction ofinflammation. Such agents may ameliorate symptoms or improve outcome viaa similar mechanism of action, e.g., anti-TGFβ antibodies, or by adifferent mechanism, e.g., interferon-γ. Such agents may additionallyameliorate symptoms directly or indirectly associated with aCTGF-associated disorder or a predisposition to develop aCTGF-associated disorder, e.g., angiotensin-converting enzyme (ACE)inhibitors and angiotensin receptor blockers (Arbs).

For example, scleroderma patients receiving infusions of the stableprostacyclin agonist Iloprost frequently report an improvement in skintightness consistent with an inhibitory effect on scar tissue formationby skin fibroblasts. Prostanoids have been shown to exert an inhibitoryeffect on collagen synthesis, and several lines of evidence demonstratethat Iloprost blocks CTGF induction in scleroderma. (Korn et al. (1980)J Clin Invest 65:543-554; Goldstein and Polger (1982) J Biol Chem257:8630-8633; and Stratton et al. (2001) J Clin Invest 108:241-250.)CTGF is elevated seven-fold in blister fluid in patients withscleroderma compared with healthy controls, however patients receivingintravenous administration of Iloprost show a marked decrease in CTGF inblister fluid. (Stratton et al. (2001) J Clin Invest 108:241-250.) Takentogether, these results suggest that some of the benefits of Iloprosttherapy in scleroderma might derive from antifibrotic effects mediatedvia reduction in CTGF levels. As there are concerns regarding the use ofa potent vasodilatory and anti-platelet prostacyclin analog in chronicsystemic administration in scleroderma patients, a therapy utilizing ananti-CTGF antibody alone or in conjunction with reduced levels ofIloprost could provide a safe and effective treatment for scleroderma.

Additional Uses

The antibodies of the invention also are useful as affinity purificationagents. In this process, the antibodies against CTGF are immobilized ona suitable support, such as Sephadex resin or filter paper, usingmethods well known in the art. The immobilized antibody then iscontacted with a sample containing the CTGF to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except the CTGF thatis bound to the immobilized antibody. Finally, the support is washedwith another suitable solvent, such as glycine buffer (pH 5.0), thatwill release the CTGF from the antibody.

EXAMPLES

The invention will be further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.These examples are provided solely to illustrate the claimed invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods which are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Example 1. Production of Recombinant Human CTGF

A recombinant human CTGF baculovirus construct was produced as describedin Segarini et al. ((2001) J Biol Chem 276:40659-40667). Briefly, a CTGFcDNA comprising only the open reading frame was generated by PCR usingDB60R32 (Bradham et al. (1991) J Cell Biol 114:1285-94) as template andthe primers 5′-gctccgcccgcagtgggatccATGaccgccgcc-3′ and5′-ggatccggatccTCAtgccatgtctccgta-3′, which add BamHI restriction enzymesites to the ends of the amplified product. The native start and stopcodons are indicated in capital letters.

The resulting amplified DNA fragment was digested with BamHI, purifiedby electrophoresis on an agarose gel, and subcloned directly into theBamHI site of the baculovirus PFASTBAC1 expression plasmid (InvitrogenCorp., Carlsbad Calif.). The sequence and orientation of the expressioncassette was verified by DNA sequencing. The resulting CTGF expressioncassette was then transferred to bacmid DNA by site-specificrecombination in bacteria. This bacmid was then used to generate a fullyrecombinant CTGF baculovirus in Spodoptera frugiperda SF9 insect cellsaccording to protocols supplied by the manufacturer (BAC-TO-BACExpression System manual; Invitrogen). Expansion of recombinantbaculovirus titers in Sf9 insect cells was performed using standardprocedures known in the art.

Hi5 insect cells were adapted for suspension growth by serial passage ofcells in shake flask culture accompanied by enrichment at each passagefor separated cells. Suspension Hi5 cells were cultured in 1 L SF90011SFM media (Invitrogen) supplemented with 20 μg/ml gentamicin (Mediatech,Inc., Herndon Va.) and 1× lipid (Invitrogen) in disposable 2.8 LFernbach culture flasks (Corning Inc., Acton Mass.) on a shaker platformat 110 rpm at 27° C. Once cells reached a density of 1.0-1.5×10⁶cells/ml with a viability of >95%, they were infected with recombinantbaculovirus at a multiplicity of infection (MOI) of 10. The cultureswere then incubated at 27° C. for an additional 40 to 44 hours. Theconditioned media, which contains rhCTGF, was collected, chilled on ice,and centrifuged at 5000×g. The supernatant was then passed through a0.45 mm filter.

Alternatively, recombinant rat CTGF was produced by inserting clone2-4-7, which encodes rat CTGF (Schmidt et al., U.S. Pat. No. 6,348,329),into pMK33 expression vector (constructed by Michael Koelle, StanfordUniversity Ph.D. dissertation, 1992). The rat CTGF expression constructwas transfected into Schneider 2 cells (American Type CultureCollection, Manassas Va.; Schneider (1972) J Embryol Exp Morphol27:353-365) using CELLFECTIN reagent (Invitrogen Corp., CarlsbadCalif.). Cells were grown in media containing 300 μg/ml hygromycin B for6 weeks, and were then grown without selection for three days.Expression of CTGF was induced by the addition of 500 μM CuSO₄ and 100μM ZnSO₄, and after four days the medium was harvested and clarified bycentrifugation and filtration as above.

CTGF produced by either method described above was purified as follows.Four liters of conditioned media was loaded over a 5 ml HI-TRAP heparincolumn (Amersham Biosciences Corp., Piscataway N.J.) pre-equilibratedwith 50 mM Tris (pH7.5), 150 mM NaCl. The column was washed with 10column volumes of 350 mM NaCl, 50 mM Tris (pH 7.5). CTGF was eluted fromthe column with an increasing NaCl salt gradient. Eluted fractions werescreened by SDS-PAGE, and those containing CTGF were pooled.

Heparin purified CTGF was diluted to a final conductivity of 5.7 mS withnon-pyrogenic double-distilled water and the pH was adjusted to 8.0. AQ-SEPHAROSE strong anion exchange column (Amersham Biosciences)containing approximately 23 ml resin connected in tandem with acarboxymethyl (CM) POROS polystyrene column (Applied Biosystems)containing approximately 7 ml resin was utilized for endotoxin removal,and capture and elution of purified rhCTGF. Prior to the sample load,the tandem column was washed with 0.5 M NaOH, followed by 0.1 M NaOH,and finally equilibration buffer. The load sample was passed over thetandem column, the Q-Sepharose column was removed, and CTGF was elutedfrom the CM POROS column (Applied Biosystems) with an increasing 350 mMto 1200 mM NaCl gradient. The purity of the eluted fractions containingCTGF was evaluated by SDS-PAGE analysis before forming a final samplepool.

Example 2. CTGF N-Terminal and C-Terminal Fragment Production

N-terminal fragments and C-terminal fragments of CTGF were prepared asfollows. Recombinant human CTGF, prepared and purified as describedabove, was digested at room temperature for 6 hours by treatment withchymotrypsin beads (Sigma Chemical Co., St. Louis, Mo.) at 1.5 mg ofCTGF per unit of chymotrypsin. The mixture was centrifuged, thechymotrypsin beads were discarded, and the supernatant, containingenyzmatically-cleaved rhCTGF, was diluted 1:5 with 50 mM Tris, pH 7.5.The diluted supernatant was applied to a Hi-Trap heparin column. Theflow-through, containing N-terminal fragments of CTGF, was collected.The heparin column was washed with 350 mM NaCl, and bound C-terminalfragments of CTGF were eluted with a linear gradient of 350 mM to 1200mM NaCl, as described above. The fractions were analyzed by SDS-PAGE,and fractions containing C-terminal fragments of CTGF were pooled.

The heparin column flow-through, which contained N-terminal fragments ofCTGF, was adjusted to 0.5 M ammonium sulfate/50 mM Tris, pH 7.5 and thenloaded onto a 15 ml phenyl sepharose HP column (Amersham-Pharmacia),which had been pre-equilibrated with 0.5 M ammonium sulfate/50 mM Tris,pH 7.5. The column was washed with 15 column volumes of 0.5 M ammoniumsulfate/50 mM Tris, pH 7.5, and bound N-terminal fragments of CTGF wereeluted with a linear gradient of 0.5 M to 0 M ammonium sulfate/50 mMTris, pH 7.5, over approximately 15 column volumes. Fractions wereanalyzed by SDS-PAGE, and fractions containing N-terminal fragments ofCTGF were pooled. The pooled solution was concentrated and the bufferexchanged with 50 mM Tris, 400 mM NaCl (pH 7.2), using an UL IRACELAMICON YM10 ultrafiltration membrane (Millipore Corp., Bedford Mass.).

Example 3. Production of Human Anti-CTGF Monoclonal Antibodies

Fully human monoclonal antibodies to human CTGF were prepared usingHUMAB mouse strains HCo7, HCo12 and HCo7+HCo12 (Medarex, Inc., PrincetonN.J.). Mice were immunized by up to 10 intraperitoneal (IP) orsubcutaneous (Sc) injections of 25-50 mg recombinant human CTGF incomplete Freund's ajuvant over a 2-4 weeks period. The immune responsewas monitored by retroorbital bleeds. Plasma was screened by ELISA (asdescribed below), and mice with sufficient titers of anti-CTGFimmunogolobulin were used for fusions. Mice were boosted intravenouslywith antigen 3 and 2 days before sacrifice and removal of the spleen.

Single cell suspensions of splenic lymphocytes from immunized mice werefused to one-fourth the number of P3X63-Ag8.653 nonsecreting mousemyeloma cells (American Type Culture Collection (ATCC), Manassas Va.)with 50% PEG (Sigma, St. Louis Mo.). Cells were plated at approximately1×105 cells/well in flat bottom microtiter plate and incubated for abouttwo weeks in high-glucose DMEM (Mediatech, Herndon Va.) containingL-glutamine and sodium pyruvate, 10% fetal bovine serum, 10% P388D1(ATCC) conditioned medium, 3-5% origen (Igen International, GaithersburgMd.), 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 mg/ml gentamycin, and1× HAT (Sigma). After 1-2 weeks, cells were cultured in medium in whichthe HAT was replaced with HT. Individual wells were then screened byELISA (described below). Antibody secreting hybridomas were replated,screened again, and, if still positive for anti-CTGF antibodies, weresubcloned at least twice by limiting dilution. The stable subclones werethen cultured in vitro to generate small amounts of antibody in tissueculture medium for characterization. One clone from each hybridoma thatretained the reactivity of the parent cells was used to generate 5-10vial cell banks stored in liquid nitrogen.

ELISA assays were performed as described by Fishwild et al. (1996,Nature Biotech 14:845-851). Briefly, microtiter plates were coated with1-2 μg/ml purified recombinant CTGF in PBS at 50 μl/well, incubated at4° C. overnight, then blocked with 200 μl/well 5% chicken serum inPBS/Tween (0.05%). Dilutions of plasma from CTGF-immunized mice orhybridoma culture supernatants were added to each well and incubated for1-2 hours at ambient temperature. The plates were washed with PBS/Tweenand then incubated with a goat-anti-human IgG Fc polyclonal antibodyconjugated with horseradish peroxidase (HRP) for 1 hour at roomtemperature. After washing, the plates were developed with 0.22 mg/mlABTS substrate (Sigma) and analyzed by spectrophotometer at 415-495 nm.

Example 4: Antibody Characterization

Hybridomas that produced antibodies to human CTGF were prepared asdescribed in Example 3. Cloned hybridoma cells were grown in Dulbecco'sModified Eagle Medium-High Glucose/RPMI 1640 (50:50) with 8 mML-Glutamine, ½×Nonessential Amino Acids, and 10% Fetal Bovine Serum.Cells expanded for antibody preparation were grown in the same mediawith 1.5% Low IgG Fetal Bovine Serum for 4-9 days at 37° C. and 6% CO2.The resulting conditioned media was cleared of cells and concentratedusing a tangential flow filtering/concentrating system. The concentratewas passed over a protein-A column and bound monoclonal antibodieseluted with 100 mM glycine, pH 3. The eluate was neutralized with 1 MTris, pH 8.0, and dialyzed against PBS.

4.1 Epitope Mapping

Epitope mapping of antibodies by competitive binding experiments is wellknown by those skilled in the field of immunology. (See, e.g., Van DerGeld et al. (1999) Clinical and Experimental Immunology 118:487-96.)Each antibody population isolated from cells propagated from a uniquecloned hybridoma cell was mapped and assigned to a specific bindingdomain on human CTGF using standard binding and blocking experiments.(See, e.g., Antibodies: A Laboratory Manual (1988) Harlow and Lane(eds), Cold Spring Harbor Laboratory Press; Tietz Textbook of ClinicalChemistry, 2^(nd) ed., (1994) Chapter 10 (Immunochemical Techniques),Saunders; and Clinical Chemistry: Theory, Analysis, Correlation (1984)Chapter 10 (Immunochemical Techniques) and Chapter 11 (CompetitiveBinding Assays), C. V. Mosby, St. Louis.) Independent binding domainswere initially defined by antibody competition experiments in which twodifferent antibodies were incubated in sequential order on CTGF coatedplates. If steric hindrance from the first antibody prevented the secondantibody from binding to CTGF, then the two antibodies were assigned tothe same binding domain. It should be understood, however, that twoantibodies might have distinct epitopes yet be near enough to each otherto be designated as members of the same binding domain.

Binding domains spanning all four exons of human CTGF were identified.All of the binding domains are conformationally defined, such that theantibodies bind to CTGF under non-reducing conditions in western blotassays. Some of the antibodies also bound to CTGF under reducingconditions in western blot assays, suggesting that each of theseantibodies bound to a linear epitope on the CTGF protein. Also,antibodies representing a subset of the binding domains showcross-reactivity to mouse CTGF in western blot analysis. The antibodyfrom each group having the highest affinity for whole CTGF was used forfurther characterization and analysis.

More refined epitope mapping was performed by ELISA analysis usingspecific recombinantly expressed fragments of CTGF. For example,antibodies that recognized epitopes on the N-terminal domain of CTGFwere identified by ELISA analysis against immobilized fragments obtainedfrom recombinant expression of exon 2 and/or exon 3 of the CTGF gene. Inthis manner, antibodies that specifically recognize N-terminal domainsor N-terminal fragments of CTGF were selected and further characterized.Antibodies that specifically recognize C-terminal domains or C-terminalfragments of CTGF were also selected and further characterized.

The epitope group defined by mAb1 binds to a linear epitope on theN-terminal fragment of CTGF encoded by exon 3. A series of truncatedsynthetic peptides covering regions encoded by the polynucleotide ofexon 3 were generated, and ELISA tests using these peptides wereconducted to further define the epitope of mAb1. The results aresummarized in Table 1; a “+” indicates binding between the peptide andmAb1, whereas a “−” indicates mAb1 does not bind to the peptide. Aboldfaced italic “C” indicates a cysteine residue in the peptide thatwas essential for mAb1 binding. An underlined “C” indicates a cysteineresidue added to the end and not a part of the native CTGF sequence.

TABLE 1 mAb1 binding to truncated peptide series encoded by exon 3. SEQmAb1 ID Peptide Sequence binding NO: N-CTGF + 27 Exon 3 + 28 Pep135CPLCSMDVRLPSPDCPFPRRVKLP + 22 PC5444 PLSSMDVRLPSPDS - 33 PC5445RLPSPDSPFPRRVKLPGK + 23 PEP5 RLPSPDCPFPRRVKL + 24 P40340 RLPSPD

PFPRRV + 25 P40341 RLPSPDSPFPRRV - 34 P40342 LPSPD

PFPRRVKL + 26 10MER SPDSPFPRRV - 35 10MER2 SPDCPFPRRV - 36 9MERPDSPFPRRV - 37 9MER2 CPFPRRVKL - 38 8MER DSPFPRRV - 39 8MER2 CFPRRVKL -40 7MER CPRRVKL - 41 6MER CRRVKL - 42 5MER CRVKL - 43

Therefore, mAb1 is a member of an antibody class that binds to theN-terminal region of CTGF. The linear epitope on CTGF necessary andsufficient for binding of mAb1 is defined by amino acid residue L143through V154 of human CTGF (SEQ ID NO:2). Further confirmation of mAb1binding specificity for this peptide was obtained by RIA and affinitychromatography. Antibodies that share this epitope, in part or in whole,are specifically included in the present invention. Additionally,antibodies that compete with mAb1 for binding to CTGF or a fragmentthereof are also specifically included in the present invention.

4.2 Antibody Affinity for CTGF

Antibody affinity is defined as the strength of the total noncovalentinteractions between a single antigen-binding site on an antibody and asingle epitope on an antigen Affinity is calculated by measuring theassociation constant (K_(a)), such that

${Affinity} = {K_{a} = {\frac{\left\lbrack {{Ab} \cdot {Ag}} \right\rbrack}{\left\lbrack {Ab} \right\rbrack\left\lbrack {Ag} \right\rbrack} = \frac{1}{K_{d}}}}$

where [Ab] is the concentration of free antigen binding site on theantibody, [Ag] is the concentration of free antigen, [Ab·Ag] is theconcentration of antigen binding site on the antibody occupied byantigen, and K_(d) is the dissociation constant of the antibody-antigencomplex.

The affinity of each antibody population identified by epitope mappingwas measured using RIA, wherein whole rhCTGF was radio-iodinated andadded to wells containing immobilized monoclonal antibody, as follows.Recombinant human CTGF was radiolabeled with ¹²⁵I using the chloramine-Tmethod. (See, Greenwood et al. (1963) Biochem J 89:114-123.) Typically,at least 60% of the ¹²⁵I was incorporated and the specific activity ofthe labeled CTGF was at least 1×10⁵ cpm/ng, although labeled CTGF oflower specific activity can be used in the radioimmunoassay. Goatanti-human IgG, γFc-specific capture antibody (Jackson ImmunoResearch)in Ca²⁺- and Mg²⁺-free DPBS (Mediatech, Herndon Va.) was added to thewells of a MAXISORP BREAKAPART microtiter plate (Nalge NuncInternational, Rochester N.Y.) and allowed to bind overnight at 4° C.The wells were then blocked with 1% BSA in Ca²⁺- and Mg²⁺-free DPBS forat least 4 hours at 4° C. The blocking solution was removed and 100 μlof test antibody at 2-50 ng/ml in Ca²⁺- and Mg²⁺-free DPBS was added andallowed to bind overnight at 4° C. Mixtures of serial dilutions ofunlabelled CTGF in a constant amount of [¹²⁵I]rhCTGF were added to wellsand incubated at room temperature for 4 to 8 hours. Wells were thenwashed four times with 0.1% Tween 20 in Ca²⁺- and Mg²⁺-free PBS(Mediatech), and the wells of the microtiter plate were separated andcounted in a gamma counter.

Affinity was estimated graphically by the method of Scatchard (1948, AnnNY Acad Sci 51:660-72). The total concentration of labeled CTGF appliedto the plate was calculated as

$\left\lbrack {CTGF} \right\rbrack_{total} = {{\frac{cpm\_ applied}{{cpm}/{fmol}} \cdot \frac{1}{0.1{\_ ml}}} + \frac{\left\lbrack {CTGF} \right\rbrack_{cold\_ stock}}{dilution}}$

where cpm_applied are counts obtained from control vials, which areloaded with CTGF mixtures in parallel with the wells of the microtiterplate; cpm/fmol is the specific activity of the [¹²⁵I]CTGF,[CTGF]_(cold_stock) is the concentration of unlabelled CTGF added toeach well, and dilution is the dilution factor for the unlabelled CTGF.

The concentration of CTGF bound to antibody is calculated from theproportion of counts bound to the wells and the total concentration ofCTGF applied to the wells.

$\left\lbrack {CTGF} \right\rbrack_{bound} = {\frac{\left( {{cpm\_ bound} - {blank}} \right.}{cpm\_ total} \cdot \lbrack{CTGF}\rbrack_{total}}$

The concentration of free (unbound) CTGF is the difference between thetotal concentration of CTGF applied and the concentration of bound CTGF.

[CTGF]_(free)=[CTGF]_(total)−[CTGF]_(bound)

Scatchard plots of affinity determinations for antibodies of theinvention are shown in FIG. 2. FIG. 2A plots the binding of an antibodyof the invention, mAb2, to [¹²⁵I]rhCTGF in the presence of increasingconcentrations of unlabeled rhCTGF. FIG. 2B plots the binding of anexemplary antibody of the invention, mAb1, to [¹²⁵I]rhCTGF in thepresence of increasing concentrations of unlabeled rhCTGF. Greaterweight is given points with similar proportions of bound and unboundCTGF, because these points will have bound counts in substantial excessof the blanks (hence, well-determined bound counts), but stillsubstantially less than the total counts applied (hence, well-determinedfree counts). Maximum binding (B_(max)) and K_(d) are represented as thex-intercept and y-intercept, respectively.

The affinity (K_(d)) of mAb1 for CTGF is less than 10⁻⁹ M, the affinitytypically found in commercially successful antibody therapeutics. (See,e.g., Maini et al. (1998) Arthritis Rheum 41:1552-1563; Targan et al.(1997) N Engl J Med 337:1029-1035; Bumgardner et al. (2001)Transplantation 72:839-45; and Kovarik et al. (1999) Transplantation68:1288-94.) Thus, mAb1 is a suitable candidate for therapeutic use, andantibodies that share epitope binding with mAb1, as described above, andhave an affinity for CTGF that is similar to or greater than mAb1 (thatis, a K_(d)≤10⁻⁹) are likewise suitable candidates for therapeutic use.Antibodies sharing epitope binding with mAb1, but have lower affinity(i.e., higher K_(d)) than mAb1, are also embodied within the presentinvention and are potentially useful in various assays and diagnosticapplications as described herein. Such antibodies may additionally beuseful in therapeutic applications, especially if they have a highavidity for antigen, as described below.

4.3 Antibody Avidity

For antibodies with more than one antigen-binding site (multivalency),the affinity at one binding site does not always reflect the truestrength of the antibody-antigen interaction. When a multivalentantibody binds to an antigen having multiple repeating epitopes, theinteraction of one antigen interaction at one binding site on theantibody increases the chance of antigen interaction with the additionalbinding sites. Avidity measures the functional combining strength of anantibody with its antigen, which is related to both the affinity of thereaction between the epitopes and paratopes, and the valencies of theantibody and antigen. Thus, avidity provides a more accurate measure ofan antibody's tendency to dissociate.

High avidity can compensate for low affinity. For example, IgMantigen-binding sites are generally lower affinity than IgG, but themultivalency of IgM gives it a high avidity, thus enabling it to bindantigen effectively.

To determine the avidity of antibodies of the invention, Fab fragmentswere first prepared by conventional papain digestion of thecorresponding immunoglobulin. Immobilized Protein A was then used toseparate the Fab fragments from the Fc and undigested antibody.

Approximately 1 ml immobilized papain slurry containing 0.5 ml settledgel, 250 μg papain, and 3.5 BAEE units was washed 3×1 ml and 1×10 mlwith Digestion Buffer (DB; 20 mM sodium phosphate, 10 mM EDTA, 20 mMcysteine, pH 7.0). The slurry was then resuspended with 0.3 ml DB, mixedwith 1.1 ml antibody (approximately 5 mg, pH 7), and agitated overnightat 37° C. The antibody digest was then separated from the resin, and Fabfragments were separated from Fc fragments and undigested antibody byaffinity chromatography using Protein A. Purity of Fab fragment wasmonitored by SDS-PAGE (FIG. 3A).

Monovalent binding was distinguished from bivalent binding by elutingantigen-bound antibodies with varying concentrations of thiocyanate. Byincreasing chaotropic ion (thiocyanate) concentration in the solution,lower affinity associations (e.g., monovalent binding of Fab to antigen)are disrupted first, while higher affinity associations (e.g., bivalentbinding of IgG to ligand) remain undisturbed. Thus, by increasingthiocyanate concentration, two different bindings can be distinguished.

Plates were coated with 10 μg/ml CTGF or CTGF peptides in 50 mMbicarbonate buffer (pH 8.5) at 4° C. overnight, blocked with blockercasein/TBS at 4° C. overnight, and then incubated with 100 μg/mlantibody or corresponding Fab in blocker casein/TBS at room temperatureovernight with agitation. Plates were then incubated with dilutions(1:1) of thiocyanate (0-7.6 M) in 100 mM phosphate buffer (pH 6.0) for15 minutes at room temperature with agitation, followed by an alkalinephosphatase-mouse anti-human (Fab′) 2 conjugate (1:1000 dilution) atroom temperature for 45 minutes. Alkaline phosphatase substrate (1mg/ml; Sigma) in 1 M diethanolamine, 0.5 mM MgCl₂ (pH9.8) was added,plates were incubated at room temperature, and the absorbance at 405 nmwas determined after 2, 10, 20, and 60 minutes.

The affinity index is the concentration of chaotropic agent(thiocyanate) that produces a 50% reduction in initial absorbance. Foran exemplary antibody of the invention, mAb1, the affinity index fordissociation of Fab from CTGF was 0.46 M, whereas the affinity index fordissociation of intact IgG from CTGF was 1.8 M (FIG. 3B). Thus, mAb1binds to antigen predominantly bivalently (avidity), and dissociatesfrom antigen much more slowly than an antibody that binds monovalently.Additional antibodies of the invention, which share epitope-bindingparameters with mAb1, may be similarly bivalent or they may be mono- ormulti-valent. Any of the antibodies of the invention may be manipulatedto improve avidity, e.g., by combining epitope-binding sites into asingle antibody construct, e.g., a tribody, etc. (See, e.g., Schoonjanset al. (2000) J Immunol 165:7050-7057.)

4.4 Cross-Reactivity

The radioimmunoassay described above (Example 4.2) was used to determinecross-reactivity of the antibodies, except unlabelled rhCTGF wasreplaced with another unlabelled competitor, rat CTGF derived fromnormal rat kidney (NRK) cells. NRK cells were grown until confluent andthen the culture media was changed to serum-free media containing 2ng/ml TGF-β2, 50 μg/ml heparin, and 250 μg/ml BSA. Conditioned mediumwas collected after two days of culture, centrifuged to remove debris,and incubated with heprin-sepharose beads (1/100 v/v beadsuspension:medium) for 2 hrs at 4° C. with agitation. The mixture wasthen centrifuged; the beads were collected and washed with PBS, and thenlysed in SDS buffer.

A Scatchard plot of the binding of mAb2 to [¹²⁵I]rhCTGF in the presenceof increasing concentrations of unlabeled rat CTGF is shown in FIG. 4A;and a Scatchard plot of the binding of mAb1 to [¹²⁵I]rhCTGF in thepresence of increasing concentrations of unlabeled rat CTGF is shown inFIG. 4B. As can be seen in the figure, mAb1 binds to both human and ratCTGF, while mAb2 binds to human but does not bind to rat CTGF.

For mAb1, the Scatchard plots for competition with rat CTGF (FIG. 4B)have shallower slopes, lower apparent affinity, and higher apparentB_(max) than the plots for competition with rhCTGF (FIG. 2B). Thus,although rat CTGF is able to compete with human CTGF for binding tomAb1, the antibody has higher affinity for recombinant human CTGF thanfor recombinant rat CTGF. mAb1 also cross-reacts with mouse and monkeyCTGF (data not shown). Antibodies showing suitable affinity for CTGFfrom other species may be used in treatment and prevention of disordersin those species. For example, an antibody of the invention that shows asuitable K_(d) for canine CTGF could be used to treat a CTGF-associateddisorder in dogs. Antibodies of the invention that show cross-speciesaffinity, such as mAb1, are also useful as research tools, to studyCTGF-associated disorders in various animal models.

4.5 Glycosylation

The radioimmunoassay described above (Example 4.2) was used to determinethe effect of antibody glycosylation on antigen binding affinity.Antibody mAb1 was treated for 8 days at 37° C. in PBS, 0.5 M EDTA, pH8.0, with peptide N-glycosidase F (PNGase F), which cleavesoligosaccharides from N-linked glycoproteins. After incubation, thereaction solution was either used directly or fractionated on a proteinA-SEPHAROSE FASTFLOW column (Amersham Bioscience, Piscataway N.J.) andeluted with 0.1 M glycine-HCl, pH 2.5. Antibody recovery afterfractionation was approximately 87%, and the endotoxin level was 0.30EU/mg. Deglycosylation was confirmed by SDS-PAGE. Binding activity ofdeglycosylated antibody to human recombinant CTGF was identical withinexperimental error to the binding activity of the glycosylated form ofthe antibody.

As various cells produce different glycosylation patterns, production ofrecombinant proteins, e.g., antibodies, in cultured cells ornon-homologous species may generate non-native glycosylation. Someproteins require specific glycosylation for activity, and alteredglycosylation reduces activity; e.g., in the case of antibodies,affinity for antigen is reduced. Protein production in certain systems,e.g., plants and chicken eggs, may also produce glycosylation patternsthat are immunogenic, thus reducing the ability to use the proteins incertain applications. The ability of the present antibodies to show thesame activity in a glycosylated and non-glycosylated form demonstratethat the invention is not limited by the presence of glycosylation,particularly a species-specific glycosylation.

Example 5. Cell Migration Assay

Cell migration is a normal and important cellular event, e.g., duringdevelopment and wound healing. Cell migration is also a factor in thepathology of disorders such as formation of fibrotic lesions, and cellsisolated from fibrotic lesions are more responsive to chemotacticstimulants than cells from corresponding normal tissue.

Antibodies of the present invention were analyzed for their ability toinhibit CTGF-stimulated chemotactic migration of smooth muscle cellsusing a Boyden chamber assay as follows. Rat arterial smooth musclecells (ASMCs) in media containing 0.1% fetal calf serum (FCS) were addedto the upper compartment of a Boyden chamber, and media containingeither 300 ng/ml rhCTGF, 10% FCS, or 0.1% FCS alone was added to thelower compartment. A collagen-coated filter having pores with a diameterof 8 μm separated the upper chamber from the lower chamber. Cells wereallowed to adhere to and migrate through the filter for 2-3 hours. Thefilter was then removed, the cells on the filter were fixed and stained,and the cells that migrated through the filter were counted. Incubationwith 300 ng/ml rhCTGF increased the number of cells migrating throughthe filter approximately 5-fold relative to 0.1% FCS controls. Theincrease in migration stimulated by CTGF was approximately 27% of thechemotactic effect seen with 10% FCS, which contains multiplechemotactic factors.

The antibodies of the invention were tested for their ability to inhibitCTGF-mediated cell migration using the assay described above, excepteither anti-CTGF antibody (at 30 and 300 mg/ml) or pooled human IgG wasalso added to the lower chamber. Four fields of cells from each of 3separate filters were counted for each sample in each assay. Results areshown in Table 2.

TABLE 2 Inhibition of CTGF-mediated cell migration. Cell Migration (%)Antibody Average SD hIgG 100 10 7 (30 μg/ml) 77 13 19 (30 μg/ml) 71 1419 (300 μg/ml) 43 12

As can be seen in Table 2, antibodies that bind to CTGF within theepitope defined by mAb1 inhibit CTGF-mediated cell migration in adose-dependent manner. The antibodies of this epitope group were theonly anti-CTGF antibodies tested that repeatedly and reproduciblyinhibited CTGF-induced migration.

Various processes, such as angiogenesis, chondrogenesis, and oncogenesisrequire alterations in cell adhesion and migration. CTGF has beenassociated with both cell adhesion and migration, and the ability ofantibodies directed against CTGF to differentially affect one activityversus another provides a diverse repertoire of therapeutic agents forthe treatment of CTGF-associated conditions. The antibodies provided bythe present invention clearly demonstrate differential activity relatingto neutralization of CTGF activities. As exemplified below, theseabilities provide unique therapeutic potential in this class ofanti-CTGF antibody.

Example 6. Pulmonary Disorders

The intratracheal (IT) instillation of bleomycin in mice is a modelsystem widely used for studying lung fibrosis and for screeningpotentially desirable antifibrotic agents. Antibodies of the inventionwere tested for their ability to reduce bleomycin-induced lung fibrosisin vivo using the procedure described by Wang et al. (2000) BiochemPharmacol 60:1949-1958, as follows.

Male C57BL/6 mice were randomly divided into two groups. Mice wereanesthetized with isofluorane, and then injected intratracheally witheither a single dose of bleomycin in 0.9% saline at 0.1 unit/50 μl/mouseor 0.9% saline alone. Each group was divided and treated immediately andthereafter once every other day for a total of seven doses with eithersaline or antibody administered intraperitoneally (IP). Fourteen daysafter the IT instillation, mice were euthanized by exsanguination of thedescending abdominal aorta under anesthesia and lung tissue washarvested.

Lung collagen content was analyzed by measuring the level ofhydroxyproline and proline using the method of Palmerini et al. (1985; JChromatogr 339:285-292), except that L-azetidine 2-carboxylic acid(Aldrich) was substituted for 3,4-dehydroproline as the internalstandard. Briefly, tissue samples were hydrolysed in 6 N HCl for 22hours at 105° C. Samples underwent pre-column derivitization witho-phthaladehyde and then 4-chloro-7-nitrobenzofuran (Aldrich) to formfluorescent adducts of proline and hydroxyproline. The fluorescentadducts were separated by reverse phase HPLC followed by fluorometricdetection.

FIG. 5 shows the result of therapeutic administration of saline (SA), anexemplary antibody of the invention, mAb1, and a pool of CTGF-specificantibodies (AbsJ) were compared for their ability to suppress lungfibrosis following bleomycin treatment. As can be seen in FIG. 5A,bleomycin treatment (BL+SA) significantly increased lung hydroxyprolinecontent 168% over the control group (SA+SA; 220±15 μg/lung). However,subsequent treatment with pooled antibodies of the invention (BL+AbsJ)showed a 60% decrease in lung hydroxyproline compared to thebleomycin-treatment alone. Similarly, subsequent treatment with mAb1(BL+mAb1) showed a 70% decrease in lung hydroxyproline compared tobleomycin alone.

Histological examination of the mouse lungs revealed normal pulmonaryparenchymal tissue in the control group (not shown). In bleomycintreated lungs, however, an increase in regions of fibrosis was clearlyseen (FIG. 5B; arrows). Therapeutic administration of an antibody of theinvention subsequent to bleomycin treatment showed a clear reduction infibrosis (FIG. 5C), although some lobes still showed a mild degree ofinterstitial fibrosis. Thus, antibodies of the invention providetherapeutic benefit when administered to patients at risk for orsuffering from a pulmonary disorder such as idiopathic pulmonaryfibrosis (IPF).

Example 7. Renal Disorders 7.1. Renal Failure

Tubulointerstitial fibrosis is a major component of several kidneydiseases associated with the progression to end-stage renal failure.(Sharma et al. (1993) Kidney Int 44:774-788.) Unilateral ureteralobstruction (UUO), characterized by decreased renal function andincreased interstitial fibrosis, has been used as an experimental modelto induce tubulointerstitial damage and fibrosis. (Fern et al. (1999) JClin Invest 103:39-46.)

Mice were anesthetized with isofluorane, and then ligation of the leftureter was performed according to the method described by Moriyama etal. (1998; Kidney Int 54:110-119). Mice were treated immediatelyfollowing surgery and thereafter once every other day for a total ofseven doses with either saline or antibody administeredintraperitoneally (IP). Fourteen days after UUO, animals wereanesthetized and sacrificed by exsanguination of the descendingabdominal aorta. Both the right and left kidneys were separatelydecapsulated and weighed. Half of each kidney was fixed in 10% formalinfor histology (trichrome stain) and the other half was weighed andstored at −70° C. for hydroxyproline determination. Hydroxyproline andproline were determined as described above.

As can be seen in FIG. 6A, UUO increased the kidney collagen contentapproximately 4-fold, as measured by the hydroxyproline to proline ratioof the obstructed left kidney relative to the unobstructed right kidneyin each mouse. Treatment with an antibody of the invention, mAb1,resulted in a statistically significant dose-dependent reduction infibrosis of the obstructed kidney (FIG. 6A). However, an antibody thatbinds to a C-terminal epitope on CTGF, mAb3, did not show significanteffect. Trichrome staining of UUO kidney identifies regions of increasedcollagen accumulation (FIG. 6B, arrows), whereas treatment with anantibody of the invention shows a considerable reduction in collagenstaining in the obstructed kidney (FIG. 6C).

Alternatively, kidney fibrosis can be studied in the rat remnant kidneymodel of progressive renal failure. The model, which involves 2/3unilateral nephrectomy combined with complete renal ablationcontralaterally (5/6 total nephrectomy), induces degenerativeparenchymal changes associated with chronic renal failure in the renalremnant, and animals become uremic and exhibit marked albuminuria,glomerulosclerosis, interstitial fibrosis and tubular atrophy. (See,e.g., Frazier et al. (2000) Vet Pathol 37:328-335; and Gandhi et al.(1998) Kidney Int 54:1157-1165.)

The 5/6 nephrectomy was performed according to Frazier et al. (2000, VetPathol 37:328:335). Five-week-old male Sprague-Dawley rats (Harlan,Indianapolis Ind.) averaging 120 g were anesthetized with ketamine andxylazine, and the cranial ⅓ and caudal ⅓ of the left kidney was incised.A gauze sponge was briefly applied to provide hemostasis, the abdomenwas rinsed with saline, 0.2 ml butorphenol, and the animal was sutured.One week after the initial surgery, the contralateral kidney was removedcompletely.

Rats were divided into saline and antibody treated groups with treatmentinitiated 2 weeks following 5/6 nephrectomy. Saline or antibody at adosage of 5 mg/kg was administered by IP injection (0.5 mL each) every 3days for 15 days (a total of 5 injections). Blood and urine samples weretaken weekly from random nephrectomized rats to follow the developmentof renal disease and to correlate renal functional disturbance withhistologic changes. Results from renal fibrosis analysis, urinalysis andserum chemistry assays were compared between the groups at 18 and 28days after treatment initiation.

Renal fibrosis was evaluated independently by two pathologists in ablinded fashion; three histologic sections from each kidney wereexamined using three distinct morphologic stains: hematoxylin/eosin,Masson's trichrome and picric acid-sirius red. In addition,immunohistochemistry was performed on frozen sections to assess the typeof collagen deposition at each location in the kidney. Quantitativecollagen evaluation (hydroxyproline/proline ratio) was performed andrenal function was assessed using both urinalysis and serum chemistry ofsamples collected at the time of euthanasia.

Histologically, moderate differences in fibrosis were noted betweenuntreated and antibody treated remnant kidneys (FIG. 7). At 3 dayspost-treatment, blinded subjective evaluation resulted in a meanfibrosis score of 12.6 in saline treated group versus 10.7 in antibodytreated group (p<0.05). Statistically significant differences inhistologic fibrosis grade between antibody and saline treated rats weremaintained at 14 days post-treatment, with a mean fibrosis score of 16.9in the saline treated group versus 14.4 in the antibody treated group(p<0.05). The quantitative hydroxyproline content analysis of collagenalso demonstrated a trend towards decreased fibrosis in theantibody-treated group relative to the saline-treated group, but thedifference was not statistically significant.

Qualitative differences were also noted between treatment groups. Whilemost of the collagen deposition in the antibody treated groups waslimited to the corticomedullary and medullary interstitium, the fibrosisin the saline treated rats was multifocal to diffusely distributedthroughout the cortex and medulla. The most marked histopathologicdifferences were in the amount of glomerular fibrosis. Many of thesaline treated group had moderate to severe glomerulosclerosis withpericapsular fibrosis, thickened Bowman's membrane, synechia, andglomerular obsolescence. These changes were minimal to mild in the othergroups, including the antibody treated rat kidneys. Collagenaccumulation was visualized with Masson's trichrome and picricacid-sirius red stains.

In both models of progressive renal failure, antibody of the inventionreduced tissue degradation and improved kidney function. Thus,antibodies of the invention provide therapeutic benefit whenadministered to patients at risk for or suffering from a renal disordersuch as glomerulonephritis, IgA nephropathy, glomerulosclerosis; andkidney failure and tubule destruction due to toxins, etc.

7.2. Diabetic Nephropathy

Diabetes leads to failure of multiple organs including, but not limitedto, kidney, heart, and eye. A major component of the pathologicalprogression of diabetic organ failure is fibrosis. An established modelof diabetic nephropathy is a mouse carrying a loss-of-function mutationin the leptin receptor (Ob-R; encoded by the db gene). Key features incommon between the db/db mouse and human diabetic nephropathy includerenal hypertrophy, glomerular enlargement, albuminuria, and mesangialmatrix expansion.

Antibodies of the invention were tested using the db/db mouse model ofdiabetic nephropathy as follows. Eight-week-old db/db mice (Harlan,Indianapolis Ind.) and their heterozygous db/+ littermates were treatedby intraperitoneal injection with either antibody of the invention(CLN1; see below) or control human IgG (cIgG). In all animals, aninitial injection of 300 μg of antibody was followed by 100 μg dosesadministered 3 times per week for 60 days. Blood samples were collectedand body weights were measured at the beginning of and periodicallythroughout the treatment period. Food consumption was also recorded.

By 11 weeks, clear distinction existed between the diabetic (db/db)animals and the non-diabetic (db/+) animals with respect to body weight,blood glucose levels, and food consumption. Treatment with eitherantibody of the invention or control antibody did not significantlyaffect any of these parameters. However, various measurements of kidneyfunction demonstrated a clear difference between diabetic andnon-diabetic mice. As can be seen in Table 3, diabetic mice showedincreased kidney weight, creatinine clearance, and albumin excretionrate (AER) relative to non-diabetic mice. However, diabetic animalstreated with antibody of the invention showed normalized values for allthe parameters. All data are expressed as Mean±SEM. The number of miceper group (n) ranged from 9 to 15.

TABLE 3 Kidney function in db/db and db/+ mice. Animal Kidney wtCreatinine AER Group Treatment (mg) (ml/h) (μg/24 h) db/+ cIgG 133.8 ±5.1 2.17 ± 0.29 0.30 ± 0.02 db/+ mAb1 141.0 ± 4.3 2.37 ± 0.19 0.23 ±0.04 db/db cIgG  207.8 ± 3.9**  5.39 ± 0.36**  2.52 ± 0.20** db/db mAb1 177.4 ± 4.5*  2.76 ± 0.31^(Δ)   0.98 ± 0.09^(□) **P < 0.01 vs. db/+mice. *P < 0.01 vs. db/+ mice and P < 0.05 cIgG-treated db/db mice.^(Δ)P < 0.01 vs. cIgG-treated db/db mice. ^(□)P < 0.01 vs. db/+ mice andcIgG-treated db/db mice.

As CTGF is induced by high glucose and mediates various activitiesincluding ECM production in tissues as a result of damage, e.g., due toadvanced glycation endproduct (AGE) formation and accumulation, etc.,pathologies associated with diabetes, such as diabetic nephropathy, maybe prevented using the antibodies of the invention.

Example 8. Ocular Disorders

Increased expression of CTGF has been associated with various oculardisorders including proliferative vitreoretinopathy (PVR), maculardegeneration, and diabetic retinopathy. (See, e.g., Hinton et al. (2002)Eye 16:422-428; He et al. (2003) Arch Ophthalmol 121:1283-1288; andTikellis et al. (2004) Endocrinology 145:860-866.) The role of CTGF andthe use of anti-CTGF therapeutics has been proposed. (See InternationalPublication No. WO 03/049773.) The antibodies of the present inventionrepresent a unique, therapeutically efficacious class of anti-CTGFtherapeutic for use in such ocular disorders. The ability of antibodiesof the present invention to ameliorate complications in ocular disordersis tested in models of ocular disease as follows.

8.1. Diabetic Retinopathy

Animal models of diabetes, e.g., db/db mice, are described in Example7.2, above. Any of these models can be used to demonstrate the efficacyof treatment of diabetic retinopathy using the antibodies of theinvention. A particular model for diabetic retinopathy is providedbelow, wherein animals are injected with streptozotocin (STZ), a knowntoxin of the insulin-secreting pancreatic β-islet cells.

Diabetes is induced in rats (e.g., Long-Evans, Sprague-Dawley, etc.) byinjection, e.g., intraperitoneally, of streptozotocin (STZ), e.g., atabout 60 to 85 mg/kg body weight. To improve survival, rats may be given10% sugar water for 24 hours and/or 2 to 4 units insulin per dayfollowing STZ injection. Various factors including, e.g., body weight,urinary albumin excretion rate, blood glucose, glycated hemoglobin,blood pressure, etc., are measured after, e.g., 4, 8, and 12 weeks.Control animals injected with buffer alone are followed concurrently.One half of the STZ-treated and control rats are additionally treatedwith antibody of the invention injected, e.g., intravenously,intraperitoneally, or intraocularly. Throughout the study animals aregiven access to food and water ad libitum. Animals are sacrificed at 12weeks, and eyes are harvested and examined for histological changes.

A reduction in pathological changes in antibody-treated animals relativeto non-treated controls is indicative of therapeutic efficacy indiabetic retinopathy. As CTGF is induced by high glucose and mediatesvarious activities including ECM production in tissues as a result ofdamage, e.g., due to advanced glycation endproduct (AGE) formation andaccumulation, etc., pathologies associated with diabetes, such asdiabetic retinopathy, may be prevented using anti-CTGF therapeutics.(See, e.g., International Publication No. WO 03/049773.) The antibodiesof the present invention represent a unique, therapeutically efficaciousclass of anti-CTGF therapeutic for use in ocular disorders, e.g.,diabetic retinopathy.

8.2. PVR

Rabbit retinal pigmented epithelial (RPE) cells are isolated from adultrabbit eyes and cultured in DMEM supplemented with 10% fetal bovineserum. Subconfluent cultures (typically at passage 2 to 3) are used forall subsequent injections. At the time of injection, cultured RPE cellsare collected and suspended in PBS to approximately 2.5×10⁶ cells/ml.Approximately 0.2 mls of aqueous humor is removed from each recipientrabbit eye using a 25-gauge needle, and then RPE cells are injectedthrough the sclera to a point three millimeters posterior to the limbusjust over and above the optic disc using a 27-gauge needle. Followinginjection of RPE cells, either 0.1 ml PDGF BB (50-150 ng), CTGF (200-400ng), or PDGF and CTGF in PBS is injected through the same entrance site.The non-injected eye of each animal serves as a control. Optionally,CTGF can additionally be injected on day 7 and/or day 14 following firstinjection. One half of the animals are additionally treated withantibody of the invention injected, e.g., intravenously,intraperitoneally, or intraocularly. Depending on the administrationsite, antibody may be provided daily or administered less frequently,e.g., on days 7, 10, 14, etc.

Animals are examined using indirect ophthalmoscopic procedures tomonitor development and extent of PVR, which is classified according toparameters described by Fastenberg. (Fastenberg et al. (1982) Am JOphthalmol 93:565-572.) Animals are then sacrificed and eyes areanalyzed by histological examination for both extent of membraneformation and fibrosis. Additionally, retina and fibrotic membrane maybe collected for measurement of collagen content.

Alternatively, PVR is induced in rabbit eyes using subretinal injectionof dispase, using the model and a procedure adapted from Frenzel et al.(1998, Invest Ophthamol Vis Sci 39:2157-2164.) A subretinal bleb isformed using 50 ml (0.05 U) of Dispase (Sigma Chemical Co.) in PBS. Onehalf of the animals are additionally treated with antibody of theinvention injected, e.g., intravenously, intraperitoneally, orintraocularly. Retinal detachment is induced in approximately 75% of theinjected rabbits not receiving antibody of the invention one week aftersurgery, and in approximately 100% of these animals two weeks followingsurgery. Epiretinal membranes are examined for extent of fibrosis.

A reduction in pathological changes in antibody-treated animals relativeto non-treated controls is indicative of therapeutic efficacy in PVR. AsCTGF has been associated with tissue damage in models of PVR, anti-CTGFagents have been proposed as therapeutics for use in such disorders.(See, e.g., International Publication No. WO 03/049773.) The antibodiesof the present invention represent a unique, therapeutically efficaciousclass of anti-CTGF therapeutic for use in ocular disorders, e.g., PVR.

Example 9. Sclerosis

Sclerosis is generally characterized by diffuse fibrosis, degenerativechanges, and vascular abnormalities in the skin (scleroderma), joints,and internal organs, especially the esophagus, GI tract, lung, heart,and kidney.

9.1. Localized Granuloma Induction

Newborn mice develop a persistent localized fibrosis when administered acombination of human-derived TGF-β2 and CTGF by subcutaneous injectionover 7 consecutive days. (Mori et al. (1999) J Cell Physiol 181:153-159;Shinozaki et al. (1997) Biochem Biophys Res Commun 237:292-297.)

One day after birth, mice were divided into three treatment groups andadministered 40 μl 1% mouse serum albumin (MSA), PBS containing either800 ng TGF-β2, 400 ng CTGF, or both TGF-β2 and CTGF by subcutaneousinjection into the subscapular region for 7 consecutive days. Thecombination TGF-β2 and CTGF group was further divided into two groups,with one group additionally receiving 40 μg antibody of the invention,mAb1. On Day 11, animals were sacrificed and sections of the injectionsites were processed and stained with Mason's trichrome for histologicalassessment. The slides were randomized and evaluated qualitatively in ablinded fashion by three scientists; scoring ranged from 0 (no change)to 4 (fibrotic tissue) based on the degree of fibrosis or connectivetissue expansion (see FIG. 8). Cumulative scores from each slide fromall the individual reviewers were then calculated and the mean valuecompared among the groups using ANOVA test.

Group mean scores for the vehicle control, TGF-β2, and TGF-β2 and CTGFcombination were 0.75, 6.83 and 9.00, respectively (Table 4).

TABLE 4 Histological scoring of granuloma in neonatal mice TreatmentMean Score Std Err Group Size Vehicle 0.75 0.48 4 TGF-β2 6.83 0.65 6TGF-β2 + rhCTGF 9.00 0.72 7 TGFβ2 + CTGF + mAb1 6.17 1.40 6 TGFβ2 +CTGF + FG-3025 7.50 1.50 4 ¹ Group scoring of slides from 3 differentreaders.

Group mean score for antibody treatment was 6.17, a statisticallysignificant reduction when compared with the corresponding TGF-β2 andCTGF combination (p<0.05), while treatment with a C-terminally directed,non-neutralizing anti-CTGF antibody, mAb3, did not reduce fibrosis.Thus, antibodies of the present invention are particularly effective atreducing local sclerotic damage to tissues.

9.2. Neonatal Systemic Fibrosis

Newborn mice were divided into groups and administered dailyintraperitoneal injections for 21 consecutive days with 300 μg/kg/dayTGFβ, 300 μg/kg/day CTGF, a combination of 300 μg/kg/day each TGFβ andCTGF, or the combination of TGFβ and CTGF preceded by IP administrationof 5 mg/kg antibody of the invention, mAb1, 30 min prior to growthfactor treatment. Pups remained with their mothers during the course oftreatment. On Day 21, animals were sacrificed, major organs wereremoved, and total proline and hydroxyproline were measured as above.

Daily injections of TGFβ induced minor systemic fibrosis, whereas CTGFalone produced no response. The combination of TGFβ and CTGF inducedsystemic fibrosis with extensive collagen deposition in several organs(FIG. 10) including liver, lung, heart, GI tract, diaphragm and kidney;extensive intestinal adhesions; and a 25% increase in mortality.Administration of the antibody of the invention in conjunction withgrowth factor treatment reduced or prevented organ fibrosis (FIG. 10)and intestinal adhesions, and prevented mortality. Thus, antibodies ofthe present invention are additionally effective when administeredsystemically at reducing sclerotic damage to various tissues and organs.The results presented in Examples 10.1 and 10.2 clearly demonstrate thatthe antibodies of the invention, when administered locally orsystemically, are therapeutically efficacious for the treatment ofsclerotic conditions.

9.3. Scleroderma

The antibodies of the invention can be used to ameliorate fibrosisassociated with scleroderma. Methods that measure the extent andseverity of skin disease in scleroderma are known within the art. (See,e.g., Rodnan et al. (1979) Arthritis Rheum 22:130-40; Aghassi et al.(1995) Arch Dermatol 131:1160-1166; Brennan et al. (1982) Br J Rheumatol31:457-460; Kahaleh et al. (1986) Clin Exp Rheumatol 4:367-369; Falangaand Bucalo (1993) J Am Acad Derm 29:47-51; Seyger et al. (1997) J AmAcad Derm 37:793-796; Seyger et al. (1998) J Am Acad Dermatol 39:220-225; Black (1969) Br J Dermatol 81:661-666; Ballou et al. (1990) JRheumatol 17:790-794; and Enomoto et al. (1996) J Am Acad Dermatol35:381-387.)

For example, modified Rodnan skin score measures skin hardness using aType OO Rex DD-3 digital durometer (Rex Gauge Company, Buffalo GroveIll.) in standardized durometer units with 0.1 unit resolution.Durometer measurements are performed at all the same skin sites asmeasured by Rodnan skin scoring. Skin scores and durometer readings areperformed at baseline screening, prior to administration of an antibodyof the invention, and every three months throughout the dosing andfollow-up periods. Each measurement is repeated four times, and astructured analysis of variance and calculation of intraclasscorrelation coefficients is used to determine between repetitionvariability in relation to site and patient variability. (Fleiss (1971)Psychol Bull 76:378-382.) Correlation techniques are also used to assessthe concordance between skin scores and durometer scores, both foroverall scores and for sub-group scores, at a given point in time.Lagged correlation analyses (e.g., relating durometer scores at entry toskin scores at time t+3 months, or t+6 months of treatment withantibody) is also performed. Disease activity and functional statusinformation can also be collected, including collagen synthesis data(PIIINP measurements). Reduction in symptoms and/or complications ofscleroderma as measured using any of the methods described abovedemonstrates therapeutic efficacy of the antibodies of the presentinvention.

Example 10. Osteoarthritis

Antibodies of the present invention are tested in one of the followingmodels to demonstrate therapeutic efficacy in osteoarthritis. For thefollowing examples, concentration of antibody used is in the range ofabout 0.015 to 15 mg antibody per kilogram of subject body weight; e.g.,a dosage of about 5 mg antibody per kilogram body weight, is consideredappropriate.

Animals, e.g., 12-week-old male C57BL/6 mice, are housed in standardcages and fed a standard diet with tap water ad libitum.

10.1 Intra-Articular Injection of AdCTGF in Murine Knee Joints

A CTGF-containing adenovirus expression vector construct (AdCTGF) isprepared using the ADEASY system (Qbiogene, Carlsbad Calif.) accordingto procedures supplied by the manufacturer. Briefly, a polynucleotideencoding full-length human CTGF is inserted using standard molecularcloning techniques into a PSHUTTLE-CMV plasmid (Qbiogene). ThepShuttle-CMV-CTGF construct is then linearized and co-transfected withPADEASY-1 plasmid (Qbiogene) into competent E. coli BJ-5183 cells byelectroporation. AdCTGF is amplified and purified using proceduresdescribed by Kim et al. (2001, J Biol Chem 276:38781-38786); an emptyadenoviral vector is used as a control. Plaque-forming units (range1.0-2.1×10¹⁰/ml) and virus particles (range 0.9-1.5×10¹²/ml) are similarfor AdCTGF and control virus.

AdCTGF or control adenovirus (1×10⁷ plaque-forming units) is injectedintra-articularly; and antibodies of the invention are administered byintra-articular, intravenous, intraperitoneal, or subcutaneousinjection. Antibody may be injected at the same time as adenoviraladministration or, alternatively, therapy may begin either before orafter injection of AdCTGF. Animals receiving control adenovirus aresimilarly injected with either anti-CTGF antibody or control antibody.Non-injected knee joints serve as controls for antibody affects.

Knee joints are isolated on various days, e.g., 1, 3, 7, 14, and/or 28days, after AdCTGF injection, decalcified for 14 days inEDTA/polyvinylpyrrolidone, and stored at −20° C. using the proceduredescribed in Stoop et al. (2001, Osteoarthritis Cartilage 9:308-315).Histology of joints is analyzed to measure synovial thickness andproteoglycan depletion; in situ hybridization and immunohistochemistryis performed to identify CTGF expression, as well as expression ofadditional factors including collagen (type I and/or III), etc. Synovialfluid is collected to determine levels of CTGF, metalloproteinases, etc.Efficacy of therapy using anti-CTGF antibodies is confirmed by areduction in parameters associated with osteoarthritis relative toanimals injected with AdCTGF and treated with control antibody.

10.2 Intra-Articular Injection of AdTGFβ in Murine Knee Joints

Alternatively, antibodies may be tested in the animal model ofosteoarthritis described by Bakker et al. (2001, OsteoarthritisCartilage 9:128-136). For example, antibody of the invention or controlantibody may be injected at the same time as, subsequent to, or inadvance of intra-articular injection of 1×10⁷ pfu TGFβ-expressingadenovirus construct (AdTGFβ). Non-injected knee joints serve ascontrols for antibody effects. On various days, e.g., Day 3, 7, 14,etc., animals from each group are sacrificed and tissues are isolatedand processed. Histology of joints is analyzed to measure synovialthickness, proteoglycan depletion, and osteophyte formation, etc.; insitu hybridization and immunohistochemistry is performed to identifyCTGF expression, as well as expression of additional factors includingcollagen (type I and/or III), etc. Synovial fluid is collected todetermine levels of TGFβ, CTGF, metalloproteinases, etc. Efficacy oftherapy using antibodies of the invention is confirmed by a reduction inparameters associated with osteoarthritis relative to animals injectedwith AdTGFβ and treated with control antibody.

10.3 Intra-Articular Injection of Papain in Murine Knee Joints

Alternatively, antibodies may be tested using the procedure described invan der Kraan et al. (1989, Am J Pathol 135:1001-1014). Intra-articularinjection of papain induces osteophyte formation, fibrosis, andproteoglycan depletion from articular cartilage. The papain model isinitiated by injecting 1 unit of papain solution (Sigma, St. Louis, Mo.)into the right knee joint of the mice. The left knee joint of eachanimal serves as an internal control. Antibodies of the invention areadministered by intra-articular, intravenous, intraperitoneal, orsubcutaneous injection at the same time as, subsequent to, or in advanceof intra-articular injection of papain (0.5%/knee). On various days,e.g., Day 3, 7, 14, etc., animals from each group are sacrificed andtissues are isolated and processed. Histology of joints is analyzed tomeasure synovial thickness, proteoglycan depletion, and osteophyteformation, etc.; in situ hybridization and immunohistochemistry isperformed to identify CTGF expression, as well as expression ofadditional factors including collagen (type I and/or III), etc. Synovialfluid is collected to determine levels of TGFβ, CTGF,metalloproteinases, etc. Efficacy of therapy using anti-CTGF antibodiesis confirmed by a reduction in parameters associated with osteoarthritisrelative to animals injected with papain and treated with controlantibody.

Example 11. Cloning and Expression

Although the following example illustrates the cloning and expression ofone particular antibody of the invention, the methods are generallyapplicable to all of the antibodies described and claimed herein.

An exemplary antibody of the invention, mAb1, was first identified aspart of a complex human antibody secreted by a hybridoma cell line(8C12-F10; prepared as described in Example 3).

11.1 Cloning and Sequencing of mAb1 Heavy Chain

Messenger RNA was isolated from a culture of 8C12-F10 cells using aMICRO-FAST TRACK kit (Invitrogen) following protocols provided by themanufacturer. Two cDNA pools were then produced by second strandsynthesis using a cDNA cell cycle kit (Invitrogen) following protocolsprovided by the manufacturer and one of the following heavy chainantisense primers:

AB90 (TGCCAGGGGGAAGACCGATGG; SEQ ID NO: 3) m19 H1504R(GCTGGGCGCCCGGGAAGTATGTA; SEQ ID NO: 4)

Heavy chain variable region sequences were cloned by PCR amplificationof the AB90-primed cDNA pool using AB90 primer and one of a series ofV-region primers, including primers corresponding to conserved secretorysignal sequences, which encode the 5′ end of the respective codingregions, and framework region 1 sequences, which encode the beginning ofthe mature immunoglobulins. Pfu DNA polymerase (Stratagene) was usedaccording to recommended manufacturer's protocols, with the followingvariations: Reactions were typically carried out in 50 μl total volume,containing 1 μl cDNA, 0.75 μM each forward and reverse primer, 200 μMeach dNTP, and 1 μl Pfu polymerase (2.5 units per μl). A countdownthermal cycler program was used with an initial incubation at 94° C. for2 min prior to addition of enzyme. The following cycle parameters werethen used: Ten cycles of 94° C. for 45 seconds, 65° C. for 45 seconds,and 72° C. for 1 minute; thirty cycles of 94° C. for 45 seconds, 55° C.for 45 seconds, and 72° C. for 1 minute; and then one cycle at 72° C.for ten 10 minutes.

Only one heavy chain signal sequence primer, AB87 (ATGGAGTTTGGRCTGAGCTG;SEQ ID NO:5), which binds to VH3 family heavy chain V regions, producedsignificant product. The 453 nucleotide PCR product was cloned into PCRBLUNT II-TOPO vector (Invitrogen), clones were screened for the correctinsert size, and three clones corresponding to the PCR products weresequenced. Identical sequences were obtained for all three clones.

Heavy chain constant and UTR region sequences were cloned by PCRamplification of the m19 H1504R-primed cDNA pool. A 601 nucleotide PCRfragment corresponding to the 5′ end of the heavy chain segment wasamplified using sense primer VH3-33 29-51F (CGGCGGTGTTTCCATTCGGTGAT; SEQID NO:6) and heavy chain constant region antisense primer m19 H553R(GGGCGCCTGAGTTCCACGACAC; SEQ ID NO:7). Topoisomerase-mediated cloningwas used to clone the PCR products into PCR-BLUNT II vector(Invitrogen), as directed by the manufacturer, and the insert was thensequenced. Similarly, a 505 nucleotide PCR fragment was amplified usingsense primer m19 H439F (GTCTTCCCCCTGGCACCCTCCTC; SEQ ID NO:8) andantisense primer m19 H943R (CCCGCGGCTTTGTCTTGGCATTAT; SEQ ID NO:9), anda 503 nucleotide PCR fragment was amplified using sense primer m19H1002F (CTGGCTGAATGGCAAGGAGTA; SEQ ID NO:10) and antisense primer m19H1504R. Both fragments were separately cloned into PCR-BLUNT II vector(Invitrogen) and sequenced as described above. A fourth heavy chain PCRfragment of 586 nucleotides was amplified using sense primer m19 H645F(GGGCACCCAGACCTACATC; SEQ ID NO:11) and antisense primer m19 H1230R(CTCCGGCTGCCCATTGCTCTCC; SEQ ID NO:12) and sequenced directly.

FIG. 11A shows a diagram of the alignment of the cloned PCR fragmentswhich provided the full length nucleotide sequence (SEQ ID NO:13) thatencodes the mAb1 heavy chain (SEQ ID NO:14). The amino acid sequence ofthe heavy chain variable region most closely resembles VH3 germ linegene DP-44. Although it was not possible to tell which D segment hadbeen used, the sequence of mAb1 most closely resembles the DH4 family.The JH region most closely matches germ line JH4 and JH5. The heavychain constant region of mAb1 matches GenBank Accession No. BC016381,indicating an allotype of G1m(3).

11.2 Cloning and Sequencing of mAb1 Light Chain

Messenger RNA was isolated from a culture of 8C12-F10 cells using aMICRO-FAST TRACK kit (Invitrogen) following protocols provided by themanufacturer. Two cDNA pools were then produced by second strandsynthesis using a cDNA cell cycle kit (Invitrogen) following protocolsprovided by the manufacturer and one of the following light chainantisense cDNA primers:

AB16 (CGGGAAGATGAAGACAGATG; SEQ ID NO: 15) Ck-760R(AAGGATGGGAGGGGGTCAGG; SEQ ID NO: 16)

Light chain variable region sequences were cloned by PCR amplificationof the AB16-primed cDNA pool using AB16 primer and one of a series ofV-region primers including primers corresponding to conserved secretorysignal sequences, which encode the 5′ end of the respective codingregions, and framework region 1 sequences, which encode the beginning ofthe mature immunoglobulins. Pfu DNA polymerase (Stratagene) was usedaccording to recommended manufacturer's protocols with the variationsand cycle parameters described above.

Only one light chain signal sequence primer, AB123(CCCGCTCAGCTCCTGGGGCTCCTG; SEQ ID NO:17), which binds to VK1 familyheavy chain V regions, produced significant product. The 408 nucleotidePCR product was cloned into PCR BLUNT II-TOPO vector (Invitrogen),clones were screened for the correct insert size, and three clonescorresponding to the PCR products were sequenced. Identical sequenceswere obtained for all three clones.

Light chain constant region sequences were cloned by PCR amplificationof the Ck-760R-primed cDNA pool. The entire coding sequence and 5′ UTRregion of the light chain was amplified using light chain sense primerL15 22m (TCAGWCYCAGTCAGGACACAGC; SEQ ID NO:18) and Ck-760R. The 788nucleotide fragment was cloned into PCR BLUNT II vector (Invitrogen) andsequenced. The resulting plasmid was designated 41m6.

FIG. 11B shows a diagram of the alignment of the cloned PCR fragmentswhich provided the full length nucleotide sequence (SEQ ID NO:19) thatencodes the mAb1 light chain (SEQ ID NO:20). The amino acid sequence ofthe light chain variable region most closely matches regions encoded bygerm line Vk L15 and Jk2 nucleotide sequences. The light chain constantregion of mAb1 is identical to the reported human germ-line kappa lightchain immunoglobulin gene sequence. (Whitehurst et al. (1992) NucleicAcids Res 20:4929-4930.)

11.3 Production of mAb1 Heavy and Light Chain Expression Constructs

Full-length mAb1 heavy chain cDNA was generated by overlap extension PCRin two steps from the heavy chain PCR products described above and shownin FIG. 11A. The two 5′ PCR products were combined with the distalprimers VH3-33 29-51F and m19 H943R in a PCR overlap extension reactionto produce a single fragment of 991 nucleotides. Similarly, the two 3′PCR products were combined with the distal primers VH3-33 29-51F and m19H943R in a PCR overlap extension reaction to produce a fragment of 860nucleotides. These two PCR extension reaction products were thengel-purified and amplified together using the distal primers VH3-3329-51F and m19 H1504R to generate the 1407 nucleotide cDNA (residues 441through 1847 of SEQ ID NO:13) coding sequence of the full-length mAb1heavy chain.

The heavy chain cDNA was then cloned into PCR-BLUNT II TOPO vector(Invitrogen) to produce plasmid 43a4. The mAb1 heavy chain coding regionwas then subcloned by digestion of plasmid 43a4 with BamHI and XbaIrestriction endonucleases, followed by ligation of the excised insertinto PCDNA5-FRT expression vector (Invitrogen), which had beenpre-digested with BamHI and Nhe restriction endonucleases. The insert ofthe resulting expression plasmid, 44a1, was sequence verified beforebeing similarly subcloned in reverse orientation into PBK-CMV vector(Clontech) to produce plasmid 47a4, and into pCEP-Pu vector (E.Kohfeldt, Max-Planck-Institut fur Biochemie), a vector derived frompCEP4 vector (Invitrogen), to produce plasmid 49a1.

The 708 nucleotide cDNA (residue 415 through 1122 of SEQ ID NO:19)encoding full-length mAb1 light chain was excised from plasmid 41m6,described above, using HindIII and Xho I restriction endonucleases, andligated into PCDNA5-FRT vector (Invitrogen), which had been pre-digestedwith HindIII and XhoI restriction endonucleases, to produce themammalian expression plasmid 42b2. The insert of plasmid 42b2 wassequence verified before being similarly subcloned in reverseorientation into PBK-CMV vector (Clontech) to produce plasmid 47b3, andinto pCEP-Pu vector (E. Kohfeldt, Max-Planck-Institut fur Biochemie) toproduce plasmid 49b1.

11.4 Transfection and Expression of Antibody Chain Constructs

COS7 cells were transfected with plasmids 44a1 (mAb1 heavy chain) and42b2 (mAb1 light chain) in both separate and co-transfections usingstandard procedures. Conditioned culture media was assayed for thepresence of antibody as described in Example 4 (supra). Only medium fromcells co-transfected with both 44a1 and 42b2 expressed human antibodyhaving CTGF-binding activity as measured by ELISA using procedures asdescribed above. The antibody, herein identified as CLN1, produced bythe co-transfected COS7 cells, binds to the N-terminal half of CTGF withan affinity of 0.8 nM.

CLN1 has also been expressed in genetically modified Chinese HamsterOvary (CHO) cells. A CHO cell line expressing exemplary antibody CLN1was deposited with the American Type Culture Collection (Manassas Va.)on 19 May 2004 and is identified by ATCC Accession No. PTA-6006. Celllines can be optimized and antibody expression can be enhanced usingvarious techniques known in the art, e.g., by gene amplification asdescribed by Wigler et al. (1980; Proc Natl Acad Sci USA 77:3567-3570)with modifications as described by Ringold et al. (1981; J Mol ApplGenet 1:165-175), Gasser et al. (1982; Proc Natl Acad Sci USA79:6522-6526), and Kaufman et al. (1985; Mol Cell Biol 5:1750-1759).

Example 12: Interaction of CTGF with TGFβ

Antibodies of the invention specifically bind to regions of CTGF definedby residues encoded by exon 3 (FIG. 1B; nucleotide 418 to nucleotide 669of SEQ ID NO:1). This region encompasses amino acid 97 to amino acid 180of SEQ ID NO:2, and includes the von Willebrand Type C domain (aminoacid 103 to amino acid 164 of SEQ ID NO:2) and the epitope of mAb1(amino acid 134 to amino acid 158 of SEQ ID NO:2). Abreu et al. (2002,Nat Cell Biol 4:599-604) report that a domain corresponding to the VWCdomain of CTGF is important for interaction between CTGF and TGFβ orBMP-4, and that said interaction modulates the activity of TGFβ andBMP-4. The following experiments demonstrate that regions encoded byexon 3 are necessary and sufficient for binding of CTGF to TGFβ, andthat antibodies of the invention can block the interaction between CTGFand TGFβ.

Interaction between CTGF and TGFβ was assayed using the followingprocedure. The wells of a 96-well MAXISORP ELISA plate (Nalge Nunc) werecoated overnight at 4° C. with 10 μg/ml of either CTGF, the CTGFfragment encoded by exon 3, or the CTGF fragment encoded by exon 5, inPBS; or with PBS alone. All wells were then blocked with 1% BSA in PBS,followed by incubation for 1 hour at room temperature in 50 μl solutioncontaining TGFβ at 0, 1, 3.3, 10, 33, 100, 333, or 1000 ng/ml, andMAB612 or MAB1835 mouse anti-TGFβ monoclonal antibody (R&D Systems,Minneapolis Minn.) at 100, 300, or 1000 ng/ml in PBS, 0.05% Tween-20.MAB1835 recognizes bovine, mouse, and human TGF-β1, and -β2, and blocksbinding of TGFβ to mouse thymocytes. MAB612 recognizes TGF-β2, but doesnot inhibit TGFβ activities. Wells were washed with PBS, 0.05% Tween-20,and then incubated for 1 hour at room temperature in a solutioncontaining an alkaline phosphatase-conjugated goat anti-mouse IgGantibody diluted in PBS, 0.05% Tween-20. Plates were again washed, andp-nitrophenyl phosphate (PNPP) in 1 M ethanolamine, 1 mM MgSO4, pH 9.8was added, wells were incubated for a suitable time to develop, and thereaction was then terminated by addition of NaOH. The absorbance at λ of405 nm was measured using a spectrophotometer.

FIG. 12 shows that CTGF and the CTGF fragment encoded by exon 3 arecapable of interacting with TGFβ to an equivalent degree, whereas theCTGF fragment encoded by exon 5 did not show any binding activity towardTGFβ. Interestingly, the anti-TGFβ antibody MAB612 was able to detectCTGF-bound TGFβ in a dose-dependent manner, but the neutralizingantibody, MAB1835, was not able to detect CTGF-bound TGFβ at anyconcentration tested (data not shown). This suggests that CTGF competeswith MAB1835 for binding to TGFβ.

Anti-CTGF antibodies were tested for their ability to block bindingbetween CTGF and TGFβ. As shown in FIG. 12, anti-bodies of theinvention, exemplified by mAb4 and mAb1, blocked TGFβ binding of bothCTGF and the CTGF fragment encoded by exon 3, whereas an anti-CTGFantibody directed to a C-terminal fragment of CTGF did not blockbinding. These results provide support for a mechanism of action whereinantibodies of the invention specifically block an interaction betweenCTGF and TGFβ, and potentially between CTGF and other members of theTGFβ superfamily.

Various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims.

All references cited herein are hereby incorporated by reference intheir entirety.

1-101. (canceled)
 102. A method for treating a connective tissue growthfactor (CTGF)-associated disorder or condition comprising administeringto a subject in need thereof an effective amount of an antibody orfragment thereof, wherein the antibody, or fragment binds to a region ofhuman connective tissue growth factor (CTGF) set forth as amino acids143 to 154 of SEQ ID NO:2, thereby treating the CTGF-associated disorderor condition.
 103. The method of claim 102, wherein the antibody is amonoclonal antibody.
 104. The method of claim 102, wherein the affinityof the antibody for CTGF is at least about 10⁻⁹ M.
 105. The method ofclaim 102, wherein the antibody is a single chain antibody.
 106. Themethod of claim 102, wherein the antibody is a humanized antibody. 107.The method of claim 102, wherein the antibody is a human antibody. 108.The method of claim 102, wherein the antibody is a chimeric antibody.109. The method of claim 102, wherein the antibody is a multivalentantibody.
 110. The method of claim 102, wherein the antibody isglycosylated.
 111. The method of claim 102, wherein the antibody isnon-glycosylated.
 112. The method of claim 102, wherein the antibodyfragment is selected from the group consisting of a Fab, a F(ab)₂, and aFIT fragment.
 113. The method of claim 102, wherein the CTGF-associateddisorder is cancer.
 114. The method of claim 113, wherein the cancer isselected from the group consisting of acute lymphoblastic leukemia,angioleimyoma, angiolipoma, breast cancer, colorectal cancer,dermatofibromas, desmoplastic cancers, fibrosarcoma, gastrointestinalcancer, glioma, glioblastoma, liver cancer, ovarian cancer, pancreaticcancer, prostate cancer and rhabdomyosarcoma.
 115. The method of claim102, wherein the CTGF-associated disorder or condition is a fibroticdisorder.
 116. The method of claim 115, wherein the fibrotic disorder isselected from the group consisting of cardiac fibrosis, interstitialfibrosis, liver fibrosis, kidney fibrosis, lung fibrosis, ocularfibrosis, osteoarthritis, scleroderma, skin fibrosis, systemic fibrosis,peridural fibrosis or peritoneal fibrosis.
 117. The method of claim 116,wherein the lung fibrosis is idiopathic pulmonary fibrosis.
 118. Themethod of claim 115, wherein the fibrotic disorder results from surgery,chemotherapy or radiation therapy.
 119. The method of claim 102, whereinthe CTGF-associated disorder or condition is arthritis, atherosclerosis,retinopathy, nephropathy, transplant rejection, hypertrophic scarring,Crohn's disease, inflammatory bowel disease, keloids or maculardegeneration.
 120. The method of claim 119, wherein the transplantrejection is the rejection of an organ selected from the groupconsisting of eye, heart, kidney, liver, lung and skin.